PRO87299 polypeptides

ABSTRACT

The present invention relates to compositions containing a novel protein and methods of using those compositions for the diagnosis and treatment of immune related diseases.

This application is a divisional of, and claims priority under 35 USC§120 to, U.S. application Ser. No. 10/371,341 filed Feb. 19, 2003, whichclaims the benefit, under 35 USC §119, of U.S. Provisional Application60/421,236 filed Oct. 25, 2002. The entire disclosures of the foregoingapplications are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to compositions and methods useful for thediagnosis and treatment of immune related diseases.

BACKGROUND OF THE INVENTION

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases,non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, etc.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen —MHC complex on an antigenpresenting cell. Helper T cells also secrete a variety of cytokines,i.e., lymphokines, which play a central role in the activation of Bcells, cytotoxic T cells and a variety of other cells which participatein the immune response.

Immune related diseases could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

CD4+ T cells are known to be important regulators of inflammation.Herein, CD4+ T cells were activated and the profile of genesdifferentially expressed upon activation was analyzed. As such, theactivation specific genes may be potential therapeutic targets. In vivoco-stimulation is necessary for a productive immune proliferativeresponse. The list of costimulatory molecules is quite extensive and itis still unclear just which co-stimulatory molecules play critical rolesin different types and stages of inflammation.

The term inflammatory bowel disorder (“IBD”) describes a group ofchronic inflammatory disorders of unknown causes in which the intestine(bowel) becomes inflamed, often causing recurring cramps or diarrhea.The prevalence of IBD in the US is estimated to be about 200 per 100,000population. Patients with IBD can be divided into two major groups,those with ulcerative colitis (“UC”) and those with Crohn's disease(“CD”).

In patients with UC, there is an inflammatory reaction primarilyinvolving the colonic mucosa. The inflammation is typically uniform andcontinuous with no intervening areas of normal mucosa. Surface mucosalcells as well as crypt epithelium and submucosa are involved in aninflammatory reaction with neutrophil infiltration. Ultimately, thissituation typically progresses to epithelial damage with loss ofepithelial cells resulting in multiple ulcerations, fibrosis, dysplasiaand longitudinal retraction of the colon.

CD differs from UC in that the inflammation extends through all layersof the intestinal wall and involves mesentery as well as lymph nodes. CDmay affect any part of the alimentary canal from mouth to anus. Thedisease is often discontinuous, i.e., severely diseased segments ofbowel are separated from apparently disease-free areas. In CD, the bowelwall also thickens which can lead to obstructions. In addition, fistulasand fissures are not uncommon.

Clinically, IBD is characterized by diverse manifestations oftenresulting in a chronic, unpredictable course. Bloody diarrhea andabdominal pain are often accompanied by fever and weight loss. Anemia isnot uncommon, as is severe fatigue. Joint manifestations ranging fromarthralgia to acute arthritis as well as abnormalities in liver functionare commonly associated with IBD. Patients with IBD also have anincreased risk of colon carcinomas compared to the general population.During acute “attacks” of IBD, work and other normal activity areusually impossible, and often a patient is hospitalized.

Although the cause of IBD remains unknown, several factors such asgenetic, infectious and immunologic susceptibility have been implicated.IBD is much more common in Caucasians, especially those of Jewishdescent. The chronic inflammatory nature of the condition has promptedan intense search for a possible infectious cause. Although agents havebeen found which stimulate acute inflammation, none has been found tocause the chronic inflammation associated with IBD. The hypothesis thatIBD is an autoimmune disease is supported by the previously mentionedextraintestinal manifestation of IBD as joint arthritis, and the knownpositive response to IBD by treatment with therapeutic agents such asadrenal glucocorticoids, cyclosporine and azathioprine, which are knownto suppress immune response. In addition, the GI tract, more than anyother organ of the body, is continuously exposed to potential antigenicsubstances such as proteins from food, bacterial byproducts (LPS), etc.

Further, the risk of colon cancer is highly elevated in patients withsevere ulcerative colitis, particularly if the disease has existed forseveral years. About 20-25% of patients with IBD eventually requiresurgery for removal of the colon because of massive bleeding, chronicdebilitating illness, performation of the colon, or risk of cancer.Surgery is also sometimes performed when other forms of medicaltreatment fail or when the side effects of steroids or other medicationsthreaten the patient's health. As surgery is invasive and drasticallylife altering, it is not a highly desireable treatment regimen, and istypically the treatment of last resort. In order to better understandthis disease and possibly treat it, experiments determined that a genewas upregulated both in CD and UC when compared to normal tissue.

Despite the above identified advances in immune disorder research, thereis a great need for additional diagnostic and therapeutic agents capableof detecting the presence of a immune disorders in a mammal and foreffectively reducing these disorders. Accordingly, it is an objective ofthe present invention to identify and characterize a polypeptide that isoverexpressed in various immune cells, involved in various immunedisorders and to use that polypeptide, and the encoding nucleic acids,to produce compositions of matter useful in the therapeutic treatmentand diagnostic detection of immune disorders in mammals.

SUMMARY OF THE INVENTION A. Embodiments

The present invention concerns compositions and methods useful for thediagnosis and treatment of immune related disease in mammals, includinghumans. The present invention is based on the identification of proteins(including agonist and antagonist antibodies) which are a result ofstimulation of the immune response in mammals. Immune related diseasescan be treated by suppressing or enhancing the immune response.Molecules that enhance the immune response stimulate or potentiate theimmune response to an antigen. Molecules which stimulate the immuneresponse can be used therapeutically where enhancement of the immuneresponse would be beneficial. Alternatively, molecules that suppress theimmune response attenuate or reduce the immune response to an antigen(e.g., neutralizing antibodies) can be used therapeutically whereattenuation of the immune response would be beneficial (e.g.,inflammation). Accordingly, the PRO87299 polypeptides, agonists andantagonists thereof are also useful to prepare medicines and medicamentsfor the treatment of immune-related and inflammatory diseases. In aspecific aspect, such medicines and medicaments comprise atherapeutically effective amount of a PRO87299 polypeptide, agonist orantagonist thereof with a pharmaceutically acceptable carrier.Preferably, the admixture is sterile.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists to a PRO87299 polypeptide which comprisescontacting the PRO87299 polypeptide with a candidate molecule andmonitoring a biological activity mediated by said PRO87299 polypeptide.Preferably, the PRO87299 polypeptide is a native sequence PRO87299polypeptide. In a specific aspect, the PRO87299 agonist or antagonist isan anti-PRO87299 antibody.

In another embodiment, the invention concerns a composition of mattercomprising a PRO87299 polypeptide or an agonist or antagonist antibodywhich binds the polypeptide in admixture with a carrier or excipient. Inone aspect, the composition comprises a therapeutically effective amountof the polypeptide or antibody. In another aspect, when the compositioncomprises an immune stimulating molecule, the composition is useful for:(a) increasing infiltration of inflammatory cells into a tissue of amammal in need thereof, (b) stimulating or enhancing an immune responsein a mammal in need thereof, (c) increasing the proliferation of immunecells in a mammal in need thereof in response to an antigen, (d)stimulating the activity of immune cells or (e) increasing the vascularpermeability. In a further aspect, when the composition comprises animmune inhibiting molecule, the composition is useful for: (a)decreasing infiltration of inflammatory cells into a tissue of a mammalin need thereof, (b) inhibiting or reducing an immune response in amammal in need thereof, (c) decreasing the activity of immune cells or(d) decreasing the proliferation of immune cells in a mammal in needthereof in response to an antigen. In another aspect, the compositioncomprises a further active ingredient, which may, for example, be afurther antibody or a cytotoxic or chemotherapeutic agent. Preferably,the composition is sterile.

In another embodiment, the invention concerns a method of treating animmune related disorder in a mammal in need thereof, comprisingadministering to the mammal an effective amount of a PRO87299polypeptide, an agonist thereof, or an antagonist thereto. In apreferred aspect, the immune related disorder is selected from the groupconsisting of: systemic lupus erythematosis, rheumatoid arthritis,osteoarthritis, juvenile chronic arthritis, spondyloarthropathies,systemic sclerosis, idiopathic inflammatory myopathies, Sjögren'ssyndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia,autoimmune thrombocytopenia, thyroiditis, diabetes mellitus,immune-mediated renal disease, demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barré syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious, autoimmune chronic active hepatitis, primary biliarycirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory bowel disease, gluten-sensitive enteropathy, and Whipple'sdisease, autoimmune or immune-mediated skin diseases including bullousskin diseases, erythema multiforme and contact dermatitis, psoriasis,allergic diseases such as asthma, allergic rhinitis, atopic dermatitis,food hypersensitivity and urticaria, immunologic diseases of the lungsuch as eosinophilic pneumonias, idiopathic pulmonary fibrosis andhypersensitivity pneumonitis, transplantation associated diseasesincluding graft rejection and graft-versus-host-disease.

In another embodiment, the invention provides an antibody whichspecifically binds to any of the above or below described polypeptides.Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody. In one aspect, the presentinvention concerns an isolated antibody which binds a PRO87299polypeptide. In another aspect, the antibody mimics the activity of aPRO87299 polypeptide (an agonist antibody) or conversely the antibodyinhibits or neutralizes the activity of a PRO87299 polypeptide (anantagonist antibody). In another aspect, the antibody is a monoclonalantibody, which preferably has nonhuman complementarity determiningregion (CDR) residues and human framework region (FR) residues. Theantibody may be labeled and may be immobilized on a solid support. In afurther aspect, the antibody is an antibody fragment, a monoclonalantibody, a single-chain antibody, or an anti-idiotypic antibody.

In yet another embodiment, the present invention provides a compositioncomprising an anti-PRO87299 antibody in admixture with apharmaceutically acceptable carrier. In one aspect, the compositioncomprises a therapeutically effective amount of the antibody.Preferably, the composition is sterile. The composition may beadministered in the form of a liquid pharmaceutical formulation, whichmay be preserved to achieve extended storage stability. Alternatively,the antibody is a monoclonal antibody, an antibody fragment, a humanizedantibody, or a single-chain antibody.

In a further embodiment, the invention concerns an article ofmanufacture, comprising:

(a) a composition of matter comprising a PRO87299 polypeptide or agonistor antagonist thereof;

(b) a container containing said composition; and

(c) a label affixed to said container, or a package insert included insaid container referring to the use of said PRO87299 polypeptide oragonist or antagonist thereof in the treatment of an immune relateddisease. The composition may comprise a therapeutically effective amountof the PRO87299 polypeptide or the agonist or antagonist thereof.

In yet another embodiment, the present invention concerns a method ofdiagnosing an immune related disease in a mammal, comprising detectingthe level of expression of a gene encoding a PRO87299 polypeptide (a) ina test sample of tissue cells obtained from the mammal, and (b) in acontrol sample of known normal tissue cells of the same cell type,wherein a higher or lower expression level in the test sample ascompared to the control sample indicates the presence of immune relateddisease in the mammal from which the test tissue cells were obtained.

In another embodiment, the present invention concerns a method ofdiagnosing an immune disease in a mammal, comprising (a) contacting ananti-PRO87299 antibody with a test sample of tissue cells obtained fromthe mammal, and (b) detecting the formation of a complex between theantibody and a PRO87299 polypeptide, in the test sample; wherein theformation of said complex is indicative of the presence or absence ofsaid disease. The detection may be qualitative or quantitative, and maybe performed in comparison with monitoring the complex formation in acontrol sample of known normal tissue cells of the same cell type. Alarger quantity of complexes formed in the test sample indicates thepresence or absence of an immune disease in the mammal from which thetest tissue cells were obtained. The antibody preferably carries adetectable label. Complex formation can be monitored, for example, bylight microscopy, flow cytometry, fluorimetry, or other techniques knownin the art. The test sample is usually obtained from an individualsuspected of having a deficiency or abnormality of the immune system.

In another embodiment, the invention provides a method for determiningthe presence of a PRO87299 polypeptide in a sample comprising exposing atest sample of cells suspected of containing the PRO87299 polypeptide toan anti-PRO87299 antibody and determining the binding of said antibodyto said cell sample. In a specific aspect, the sample comprises a cellsuspected of containing the PRO87299 polypeptide and the antibody bindsto the cell. The antibody is preferably detectably labeled and/or boundto a solid support.

In another embodiment, the present invention concerns an immune-relateddisease diagnostic kit, comprising an anti-PRO87299 antibody and acarrier in suitable packaging. The kit preferably contains instructionsfor using the antibody to detect the presence of the PRO87299polypeptide. Preferably the carrier is pharmaceutically acceptable.

In another embodiment, the present invention concerns a diagnostic kit,containing an anti-PRO87299 antibody in suitable packaging. The kitpreferably contains instructions for using the antibody to detect thePRO87299 polypeptide.

In another embodiment, the invention provides a method of diagnosing animmune-related disease in a mammal which comprises detecting thepresence or absence or a PRO87299 polypeptide in a test sample of tissuecells obtained from said mammal, wherein the presence or absence of thePRO87299 polypeptide in said test sample is indicative of the presenceof an immune-related disease in said mammal.

In another embodiment, the present invention concerns a method foridentifying an agonist of a PRO87299 polypeptide comprising:

(a) contacting cells and a test compound to be screened under conditionssuitable for the induction of a cellular response normally induced by aPRO87299 polypeptide; and

(b) determining the induction of said cellular response to determine ifthe test compound is an effective agonist, wherein the induction of saidcellular response is indicative of said test compound being an effectiveagonist.

In another embodiment, the invention concerns a method for identifying acompound capable of inhibiting the activity of a PRO87299 polypeptidecomprising contacting a candidate compound with a PRO87299 polypeptideunder conditions and for a time sufficient to allow these two componentsto interact and determining whether the activity of the PRO87299polypeptide is inhibited. In a specific aspect, either the candidatecompound or the PRO87299 polypeptide is immobilized on a solid support.In another aspect, the non-immobilized component carries a detectablelabel. In a preferred aspect, this method comprises the steps of:

(a) contacting cells and a test compound to be screened in the presenceof a PRO87299 polypeptide under conditions suitable for the induction ofa cellular response normally induced by a PRO87299 polypeptide; and

(b) determining the induction of said cellular response to determine ifthe test compound is an effective antagonist.

In another embodiment, the invention provides a method for identifying acompound that inhibits the expression of a PRO87299 polypeptide in cellsthat normally express the polypeptide, wherein the method comprisescontacting the cells with a test compound and determining whether theexpression of the PRO87299 polypeptide is inhibited. In a preferredaspect, this method comprises the steps of:

(a) contacting cells and a test compound to be screened under conditionssuitable for allowing expression of the PRO87299 polypeptide; and

(b) determining the inhibition of expression of said polypeptide.

In yet another embodiment, the present invention concerns a method fortreating an immune-related disorder in a mammal that suffers therefromcomprising administering to the mammal a nucleic acid molecule thatcodes for either (a) a PRO87299 polypeptide, (b) an agonist of aPRO87299 polypeptide or (c) an antagonist of a PRO87299 polypeptide,wherein said agonist or antagonist may be an anti-PRO87299 antibody. Ina preferred embodiment, the mammal is human. In another preferredembodiment, the nucleic acid is administered via ex vivo gene therapy.In a further preferred embodiment, the nucleic acid is comprised withina vector, more preferably an adenoviral, adeno-associated viral,lentiviral or retroviral vector.

In yet another aspect, the invention provides a recombinant viralparticle comprising a viral vector consisting essentially of a promoter,nucleic acid encoding (a) a PRO87299 polypeptide, (b) an agonistpolypeptide of a PRO87299 polypeptide, or (c) an antagonist polypeptideof a PRO87299 polypeptide, and a signal sequence for cellular secretionof the polypeptide, wherein the viral vector is in association withviral structural proteins. Preferably, the signal sequence is from amammal, such as from a native PRO87299 polypeptide.

In a still further embodiment, the invention concerns an ex vivoproducer cell comprising a nucleic acid construct that expressesretroviral structural proteins and also comprises a retroviral vectorconsisting essentially of a promoter, nucleic acid encoding (a) aPRO87299 polypeptide, (b) an agonist polypeptide of a PRO87299polypeptide or (c) an antagonist polypeptide of a PRO87299 polypeptide,and a signal sequence for cellular secretion of the polypeptide, whereinsaid producer cell packages the retroviral vector in association withthe structural proteins to produce recombinant retroviral particles.

In a still further embodiment, the invention provides a method ofincreasing the activity of immune cells in a mammal comprisingadministering to said mammal (a) a PRO87299 polypeptide, (b) an agonistof a PRO87299 polypeptide, or (c) an antagonist of a PRO87299polypeptide, wherein the activity of immune cells in the mammal isincreased.

In a still further embodiment, the invention provides a method ofincreasing the proliferation of immune cells in a mammal comprisingadministering to said mammal (a) a PRO87299 polypeptide, (b) an agonistof a PRO87299 polypeptide, or (c) an antagonist of a PRO87299polypeptide, wherein the proliferation of immune cells in the mammal isincreased.

In a still further embodiment, the invention provides a method ofdecreasing the proliferation of immune cells in a mammal comprisingadministering to said mammal (a) a PRO87299 polypeptide, (b) an agonistof a PRO87299 polypeptide, or (c) an antagonist of a PRO87299polypeptide, wherein the proliferation of immune cells in the mammal isdecreased.

B. Additional Embodiments

In other embodiments of the present invention, the invention providesvectors comprising DNA encoding any of the herein describedpolypeptides. Host cell comprising any such vector are also provided. Byway of example, the host cells may be CHO cells, E. coli, or yeast. Aprocess for producing any of the herein described polypeptides isfurther provided and comprises culturing host cells under conditionssuitable for expression of the desired polypeptide and recovering thedesired polypeptide from the cell culture.

In other embodiments, the invention provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence. Example of suchchimeric molecules comprise any of the herein described polypeptidesfused to an epitope tag sequence or a Fc region of an immunoglobulin.

In another embodiment, the invention provides an antibody whichspecifically binds to any of the above or below described polypeptides.Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody.

In yet other embodiments, the invention provides oligonucleotide probesuseful for isolating genomic and cDNA nucleotide sequences or asantisense probes, wherein those probes may be derived from any of theabove or below described nucleotide sequences.

In other embodiments, the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence that encodes a PRO87299polypeptide.

In one aspect, the isolated nucleic acid molecule comprises a nucleotidesequence having at least about 80% nucleic acid sequence identity,alternatively at least about 81% nucleic acid sequence identity,alternatively at least about 82% nucleic acid sequence identity,alternatively at least about 83% nucleic acid sequence identity,alternatively at least about 84% nucleic acid sequence identity,alternatively at least about 85% nucleic acid sequence identity,alternatively at least about 86% nucleic acid sequence identity,alternatively at least about 87% nucleic acid sequence identity,alternatively at least about 88% nucleic acid sequence identity,alternatively at least about 89% nucleic acid sequence identity,alternatively at least about 90% nucleic acid sequence identity,alternatively at least about 91% nucleic acid sequence identity,alternatively at least about 92% nucleic acid sequence identity,alternatively at least about 93% nucleic acid sequence identity,alternatively at least about 94% nucleic acid sequence identity,alternatively at least about 95% nucleic acid sequence identity,alternatively at least about 96% nucleic acid sequence identity,alternatively at least about 97% nucleic acid sequence identity,alternatively at least about 98% nucleic acid sequence identity andalternatively at least about 99% nucleic acid sequence identity to (a) aDNA molecule encoding a PRO87299 polypeptide having a full-length aminoacid sequence as disclosed herein, an amino acid sequence lacking thesignal peptide as disclosed herein, an extracellular domain of atransmembrane protein, with or without the signal peptide, as disclosedherein or any other specifically defined fragment of the full-lengthamino acid sequence as disclosed herein, or (b) the complement of theDNA molecule of (a).

In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% nucleic acid sequenceidentity, alternatively at least about 81% nucleic acid sequenceidentity, alternatively at least about 82% nucleic acid sequenceidentity, alternatively at least about 83% nucleic acid sequenceidentity, alternatively at least about 84% nucleic acid sequenceidentity, alternatively at least about 85% nucleic acid sequenceidentity, alternatively at least about 86% nucleic acid sequenceidentity, alternatively at least about 87% nucleic acid sequenceidentity, alternatively at least about 88% nucleic acid sequenceidentity, alternatively at least about 89% nucleic acid sequenceidentity, alternatively at least about 90% nucleic acid sequenceidentity, alternatively at least about 91% nucleic acid sequenceidentity, alternatively at least about 92% nucleic acid sequenceidentity, alternatively at least about 93% nucleic acid sequenceidentity, alternatively at least about 94% nucleic acid sequenceidentity, alternatively at least about 95% nucleic acid sequenceidentity, alternatively at least about 96% nucleic acid sequenceidentity, alternatively at least about 97% nucleic acid sequenceidentity, alternatively at least about 98% nucleic acid sequenceidentity and alternatively at least about 99% nucleic acid sequenceidentity to (a) a DNA molecule comprising the coding sequence of afull-length PRO87299 polypeptide cDNA as disclosed herein, the codingsequence of a PRO87299 polypeptide lacking the signal peptide asdisclosed herein, the coding sequence of an extracellular domain of atransmembrane PRO87299 polypeptide, with or without the signal peptide,as disclosed herein or the coding sequence of any other specificallydefined fragment of the full-length amino acid sequence as disclosedherein, or (b) the complement of the DNA molecule of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence having at least about 80%nucleic acid sequence identity, alternatively at least about 81% nucleicacid sequence identity, alternatively at least about 82% nucleic acidsequence identity, alternatively at least about 83% nucleic acidsequence identity, alternatively at least about 84% nucleic acidsequence identity, alternatively at least about 85% nucleic acidsequence identity, alternatively at least about 86% nucleic acidsequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively at least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity to a DNA molecule that encodes the same maturepolypeptide as shown in FIG. 2 (SEQ ID NO:2).

Another aspect the invention provides an isolated nucleic acid moleculecomprising a nucleotide sequence encoding a PRO87299 polypeptide whichis either transmembrane domain-deleted or transmembranedomain-inactivated, or is complementary to such encoding nucleotidesequence, wherein the transmembrane domain(s) of such polypeptide aredisclosed herein. Therefore, soluble extracellular domains of the hereindescribed PRO87299 polypeptides are contemplated.

Another embodiment is directed to fragments of a PRO87299 polypeptidecoding sequence, or the complement thereof, that may find use as, forexample, hybridization probes, for encoding fragments of a PRO87299polypeptide that may optionally encode a polypeptide comprising abinding site for an anti-PRO87299 antibody or as antisenseoligonucleotide probes. Such nucleic acid fragments are usually at leastabout 20 nucleotides in length, alternatively at least about 30nucleotides in length, alternatively at least about 40 nucleotides inlength, alternatively at least about 50 nucleotides in length,alternatively at least about 60 nucleotides in length, alternatively atleast about 70 nucleotides in length, alternatively at least about 80nucleotides in length, alternatively at least about 90 nucleotides inlength, alternatively at least about 100 nucleotides in length,alternatively at least about 110 nucleotides in length, alternatively atleast about 120 nucleotides in length, alternatively at least about 130nucleotides in length, alternatively at least about 140 nucleotides inlength, alternatively at least about 150 nucleotides in length,alternatively at least about 160 nucleotides in length, alternatively atleast about 170 nucleotides in length, alternatively at least about 180nucleotides in length, alternatively at least about 190 nucleotides inlength, alternatively at least about 200 nucleotides in length,alternatively at least about 250 nucleotides in length, alternatively atleast about 300 nucleotides in length, alternatively at least about 350nucleotides in length, alternatively at least about 400 nucleotides inlength, alternatively at least about 450 nucleotides in length,alternatively at least about 500 nucleotides in length, alternatively atleast about 600 nucleotides in length, alternatively at least about 700nucleotides in length, alternatively at least about 800 nucleotides inlength, alternatively at least about 900 nucleotides in length andalternatively at least about 1000 nucleotides in length, wherein in thiscontext the term “about” means the referenced nucleotide sequence lengthplus or minus 10% of that referenced length. It is noted that novelfragments of a PRO87299 polypeptide-encoding nucleotide sequence may bedetermined in a routine manner by aligning the PRO87299polypeptide-encoding nucleotide sequence with other known nucleotidesequences using any of a number of well known sequence alignmentprograms and determining which PRO87299 polypeptide-encoding nucleotidesequence fragment(s) are novel. All of such PRO87299polypeptide-encoding nucleotide sequences are contemplated herein. Alsocontemplated are the PRO87299 polypeptide fragments encoded by thesenucleotide molecule fragments, preferably those PRO87299 polypeptidefragments that comprise a binding site for an anti-PRO87299 antibody.

In another embodiment, the invention provides isolated PRO87299polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified.

In a certain aspect, the invention concerns an isolated PRO87299polypeptide, comprising an amino acid sequence having at least about 80%amino acid sequence identity, alternatively at least about 81% aminoacid sequence identity, alternatively at least about 82% amino acidsequence identity, alternatively at least about 83% amino acid sequenceidentity, alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to aPRO87299 polypeptide having a full-length amino acid sequence asdisclosed herein, an amino acid sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a transmembrane protein,with or without the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein.

In a further aspect, the invention concerns an isolated PRO87299polypeptide comprising an amino acid sequence having at least about 80%amino acid sequence identity, alternatively at least about 81% aminoacid sequence identity, alternatively at least about 82% amino acidsequence identity, alternatively at least about 83% amino acid sequenceidentity, alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to anamino acid sequence shown in FIG. 2 (SEQ ID NO:2).

In a specific aspect, the invention provides an isolated PRO87299polypeptide without the N-terminal signal sequence and/or the initiatingmethionine and is encoded by a nucleotide sequence that encodes such anamino acid sequence as herein before described. Processes for producingthe same are also herein described, wherein those processes compriseculturing a host cell comprising a vector which comprises theappropriate encoding nucleic acid molecule under conditions suitable forexpression of the PRO87299 polypeptide and recovering the PRO87299polypeptide from the cell culture.

Another aspect the invention provides an isolated PRO87299 polypeptidewhich is either transmembrane domain-deleted or transmembranedomain-inactivated. Processes for producing the same are also hereindescribed, wherein those processes comprise culturing a host cellcomprising a vector which comprises the appropriate encoding nucleicacid molecule under conditions suitable for expression of the PRO87299polypeptide and recovering the PRO87299 polypeptide from the cellculture.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO87299 polypeptide as defined herein. In aparticular embodiment, the agonist or antagonist is an anti-PRO87299antibody or a small molecule.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists to a PRO87299 polypeptide which comprisecontacting the PRO87299 polypeptide with a candidate molecule andmonitoring a biological activity mediated by said PRO87299 polypeptide.Preferably, the PRO87299 polypeptide is a native PRO87299 polypeptide.

In a still further embodiment, the invention concerns a composition ofmatter comprising a PRO87299 polypeptide, or an agonist or antagonist ofa PRO87299 polypeptide as herein described, or an anti-PRO87299antibody, in combination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

Another embodiment of the present invention is directed to the use of aPRO87299 polypeptide, or an agonist or antagonist thereof as hereinbefore described, or an anti-PRO87299 antibody, for the preparation of amedicament useful in the treatment of a condition which is responsive tothe PRO87299 polypeptide, an agonist or antagonist thereof or ananti-PRO87299 antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native sequencePRO87299 cDNA, wherein SEQ ID NO:1 is a clone designated herein as“DNA332467”.

FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived from thecoding sequence of SEQ ID NO:1 shown in FIG. 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

The PRO87299 polypeptides described herein may be isolated from avariety of sources, such as from human tissue types or from anothersource, or prepared by recombinant or synthetic methods. All disclosuresin this specification which refer to the “PRO87299 polypeptide” refer toeach of the polypeptides individually as well as jointly. For example,descriptions of the preparation of, purification of, derivation of,formation of antibodies to or against, administration of, compositionscontaining, treatment of a disease with, etc., pertain to eachpolypeptide of the invention individually. The term “PRO87299polypeptide” also includes variants of the PRO87299 polypeptidesdisclosed herein.

A “native sequence PRO87299 polypeptide” comprises a polypeptide havingthe same amino acid sequence as the corresponding PRO87299 polypeptidederived from nature. Such native sequence PRO87299 polypeptides can beisolated from nature or can be produced by recombinant or syntheticmeans. The term “native sequence PRO87299 polypeptide” specificallyencompasses naturally-occurring truncated or secreted forms of thespecific PRO87299 polypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence PRO87299 polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acids sequences shown in theaccompanying figures. Start and stop codons are shown in bold font andunderlined in the figures. However, while the PRO87299 polypeptidedisclosed in the accompanying figures are shown to begin with methionineresidues designated herein as amino acid position 1 in the figures, itis conceivable and possible that other methionine residues locatedeither upstream or downstream from the amino acid position 1 in thefigures may be employed as the starting amino acid residue for thePRO87299 polypeptides.

The PRO87299 polypeptide “extracellular domain” or “ECD” refers to aform of the PRO87299 polypeptide which is essentially free of thetransmembrane and cytoplasmic domains. Ordinarily, a PRO87299polypeptide ECD will have less than 1% of such transmembrane and/orcytoplasmic domains and preferably, will have less than 0.5% of suchdomains. It will be understood that any transmembrane domains identifiedfor the PRO87299 polypeptides of the present invention are identifiedpursuant to criteria routinely employed in the art for identifying thattype of hydrophobic domain. The exact boundaries of a transmembranedomain may vary but most likely by no more than about 5 amino acids ateither end of the domain as initially identified herein. Optionally,therefore, an extracellular domain of a PRO87299 polypeptide may containfrom about 5 or fewer amino acids on either side of the transmembranedomain/extracellular domain boundary as identified in the Examples orspecification and such polypeptides, with or without the associatedsignal peptide, and nucleic acid encoding them, are contemplated by thepresent invention.

“PRO87299 polypeptide variant” means an active PRO87299 polypeptide asdefined above or below having at least about 80% amino acid sequenceidentity with a full-length native sequence PRO87299 polypeptidesequence as disclosed herein, a PRO87299 polypeptide sequence lackingthe signal peptide as disclosed herein, an extracellular domain of aPRO87299 polypeptide, with or without the signal peptide, as disclosedherein or any other fragment of a full-length PRO87299 polypeptidesequence as disclosed herein. Such PRO87299 polypeptide variantsinclude, for instance, PRO87299 polypeptides wherein one or more aminoacid residues are added, or deleted, at the N- or C-terminus of thefull-length native amino acid sequence. Ordinarily, a PRO87299polypeptide variant will have at least about 80% amino acid sequenceidentity, alternatively at least about 81% amino acid sequence identity,alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to afull-length native sequence PRO87299 polypeptide sequence as disclosedherein, a PRO87299 polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO87299 polypeptide,with or without the signal peptide, as disclosed herein or any otherspecifically defined fragment of a full-length PRO87299 polypeptidesequence as disclosed herein. Ordinarily, PRO87299 variant polypeptidesare at least about 10 amino acids in length, alternatively at leastabout 20 amino acids in length, alternatively at least about 30 aminoacids in length, alternatively at least about 40 amino acids in length,alternatively at least about 50 amino acids in length, alternatively atleast about 60 amino acids in length, alternatively at least about 70amino acids in length, alternatively at least about 80 amino acids inlength, alternatively at least about 90 amino acids in length,alternatively at least about 100 amino acids in length, alternatively atleast about 150 amino acids in length, alternatively at least about 200amino acids in length, alternatively at least about 300 amino acids inlength, or more.

“Percent (%) amino acid sequence identity” with respect to the PRO87299polypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the specific PRO87299 polypeptide sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, % aminoacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations using this method, Tables 2 and 3 demonstrate how tocalculate the % amino acid sequence identity of the amino acid sequencedesignated “Comparison Protein” to the amino acid sequence designated“PRO87299”, wherein “PRO87299” represents the amino acid sequence of ahypothetical PRO87299 polypeptide of interest, “Comparison Protein”represents the amino acid sequence of a polypeptide against which the“PRO87299” polypeptide of interest is being compared, and “X, “Y” and“Z” each represent different hypothetical amino acid residues.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, % aminoacid sequence identity values may also be obtained as described below byusing the WU-BLAST-2 computer program (Altschul et al., Methods inEnzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parametersare set to the default values. Those not set to default values, i.e.,the adjustable parameters, are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11, and scoringmatrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequenceidentity value is determined by dividing (a) the number of matchingidentical amino acid residues between the amino acid sequence of thePRO87299 polypeptide of interest having a sequence derived from thenative PRO87299 polypeptide and the comparison amino acid sequence ofinterest (i.e., the sequence against which the PRO87299 polypeptide ofinterest is being compared which may be a PRO87299 variant polypeptide)as determined by WU-BLAST-2 by (b) the total number of amino acidresidues of the PRO87299 polypeptide of interest. For example, in thestatement “a polypeptide comprising an the amino acid sequence A whichhas or having at least 80% amino acid sequence identity to the aminoacid sequence B”, the amino acid sequence A is the comparison amino acidsequence of interest and the amino acid sequence B is the amino acidsequence of the PRO87299 polypeptide of interest.

Percent amino acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison programmay be obtained from the National Institute of Health, Bethesda, Md.NCBI-BLAST2 uses several search parameters, wherein all of those searchparameters are set to default values including, for example, unmask=yes,strand=all, expected occurrences=10, minimum low complexity length=15/5,multi-pass e-value=0.01, constant for multi-pass=25, dropoff for finalgapped alignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

“PRO87299 variant polynucleotide” or “PRO87299 variant nucleic acidsequence” means a nucleic acid molecule which encodes an active PRO87299polypeptide as defined below and which has at least about 80% nucleicacid sequence identity with a nucleotide acid sequence encoding afull-length native sequence PRO87299 polypeptide sequence as disclosedherein, a full-length native sequence PRO87299 polypeptide sequencelacking the signal peptide as disclosed herein, an extracellular domainof a PRO87299 polypeptide, with or without the signal peptide, asdisclosed herein or any other fragment of a full-length PRO87299polypeptide sequence as disclosed herein. Ordinarily, a PRO87299 variantpolynucleotide will have at least about 80% nucleic acid sequenceidentity, alternatively at least about 81% nucleic acid sequenceidentity, alternatively at least about 82% nucleic acid sequenceidentity, alternatively at least about 83% nucleic acid sequenceidentity, alternatively at least about 84% nucleic acid sequenceidentity, alternatively at least about 85% nucleic acid sequenceidentity, alternatively at least about 86% nucleic acid sequenceidentity, alternatively at least about 87% nucleic acid sequenceidentity, alternatively at least about 88% nucleic acid sequenceidentity, alternatively at least about 89% nucleic acid sequenceidentity, alternatively at least about 90% nucleic acid sequenceidentity, alternatively at least about 91% nucleic acid sequenceidentity, alternatively at least about 92% nucleic acid sequenceidentity, alternatively at least about 93% nucleic acid sequenceidentity, alternatively at least about 94% nucleic acid sequenceidentity, alternatively at least about 95% nucleic acid sequenceidentity, alternatively at least about 96% nucleic acid sequenceidentity, alternatively at least about 97% nucleic acid sequenceidentity, alternatively at least about 98% nucleic acid sequenceidentity and alternatively at least about 99% nucleic acid sequenceidentity with a nucleic acid sequence encoding a full-length nativesequence PRO87299 polypeptide sequence as disclosed herein, afull-length native sequence PRO87299 polypeptide sequence lacking thesignal peptide as disclosed herein, an extracellular domain of aPRO87299 polypeptide, with or without the signal sequence, as disclosedherein or any other fragment of a full-length PRO87299 polypeptidesequence as disclosed herein. Variants do not encompass the nativenucleotide sequence.

Ordinarily, PRO87299 variant polynucleotides are at least about 30nucleotides in length, alternatively at least about 60 nucleotides inlength, alternatively at least about 90 nucleotides in length,alternatively at least about 120 nucleotides in length, alternatively atleast about 150 nucleotides in length, alternatively at least about 180nucleotides in length, alternatively at least about 210 nucleotides inlength, alternatively at least about 240 nucleotides in length,alternatively at least about 270 nucleotides in length, alternatively atleast about 300 nucleotides in length, alternatively at least about 450nucleotides in length, alternatively at least about 600 nucleotides inlength, alternatively at least about 900 nucleotides in length, or more.

“Percent (%) nucleic acid sequence identity” with respect toPRO87299-encoding nucleic acid sequences identified herein is defined asthe percentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the PRO87299 nucleic acid sequence of interest,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. For purposes herein, however, % nucleicacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “PRO87299-DNA”,wherein “PRO87299-DNA” represents a hypothetical PRO87299-encodingnucleic acid sequence of interest, “Comparison DNA” represents thenucleotide sequence of a nucleic acid molecule against which the“PRO87299-DNA” nucleic acid molecule of interest is being compared, and“N”, “L” and “V” each represent different hypothetical nucleotides.

Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, %nucleic acid sequence identity values may also be obtained as describedbelow by using the WU-BLAST-2 computer program (Altschul et al., Methodsin Enzymology 266:460-480 (1996)). Most of the WU-BLAST-2 searchparameters are set to the default values. Those not set to defaultvalues, i.e., the adjustable parameters, are set with the followingvalues: overlap span=1, overlap fraction=0.125, word threshold (T)=11,and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleicacid sequence identity value is determined by dividing (a) the number ofmatching identical nucleotides between the nucleic acid sequence of thePRO87299 polypeptide-encoding nucleic acid molecule of interest having asequence derived from the native sequence PRO87299 polypeptide-encodingnucleic acid and the comparison nucleic acid molecule of interest (i.e.,the sequence against which the PRO87299 polypeptide-encoding nucleicacid molecule of interest is being compared which may be a variantPRO87299 polynucleotide) as determined by WU-BLAST-2 by (b) the totalnumber of nucleotides of the PRO87299 polypeptide-encoding nucleic acidmolecule of interest. For example, in the statement “an isolated nucleicacid molecule comprising a nucleic acid sequence A which has or havingat least 80% nucleic acid sequence identity to the nucleic acid sequenceB”, the nucleic acid sequence A is the comparison nucleic acid moleculeof interest and the nucleic acid sequence B is the nucleic acid sequenceof the PRO87299 polypeptide-encoding nucleic acid molecule of interest.

Percent nucleic acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison programmay be obtained from the National Institute of Health, Bethesda, Md.NCBI-BLAST2 uses several search parameters, wherein all of those searchparameters are set to default values including, for example, unmask=yes,strand=all, expected occurrences=10, minimum low complexity length=15/5,multi-pass e-value=0.01, constant for multi-pass=25, dropoff for finalgapped alignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for sequence comparisons,the % nucleic acid sequence identity of a given nucleic acid sequence Cto, with, or against a given nucleic acid sequence D (which canalternatively be phrased as a given nucleic acid sequence C that has orcomprises a certain % nucleic acid sequence identity to, with, oragainst a given nucleic acid sequence D) is calculated as follows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program NCBI-BLAST2 in that program's alignment of Cand D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C.

In other embodiments, PRO87299 variant polynucleotides are nucleic acidmolecules that encode an active PRO87299 polypeptide and which arecapable of hybridizing, preferably under stringent hybridization andwash conditions, to nucleotide sequences encoding a full-length PRO87299polypeptide as disclosed herein. PRO87299 variant polypeptides may bethose that are encoded by a PRO87299 variant polynucleotide.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO87299polypeptide natural environment will not be present. Ordinarily,however, isolated polypeptide will be prepared by at least onepurification step.

An “isolated” PRO87299 polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single anti-PRO87299 monoclonal antibodies(including agonist, antagonist, and neutralizing antibodies),anti-PRO87299 antibody compositions with polyepitopic specificity,single chain anti-PRO87299 antibodies, and fragments of anti-PRO87299antibodies (see below). The term “monoclonal antibody” as used hereinrefers to an antibody obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a PRO87299 polypeptide fused to a “tagpolypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, yet is short enough suchthat it does not interfere with activity of the polypeptide to which itis fused. The tag polypeptide preferably also is fairly unique so thatthe antibody does not substantially cross-react with other epitopes.Suitable tag polypeptides generally have at least six amino acidresidues and usually between about 8 and 50 amino acid residues(preferably, between about 10 and 20 amino acid residues).

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

“Active” or “activity” for the purposes herein refers to form(s) of aPRO87299 polypeptide which retain a biological and/or an immunologicalactivity of native or naturally-occurring PRO87299, wherein “biological”activity refers to a biological function (either inhibitory orstimulatory) caused by a native or naturally-occurring PRO87299 otherthan the ability to induce the production of an antibody against anantigenic epitope possessed by a native or naturally-occurring PRO87299and an “immunological” activity refers to the ability to induce theproduction of an antibody against an antigenic epitope possessed by anative or naturally-occurring PRO87299.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native PRO87299 polypeptide disclosed herein.In a similar manner, the term “agonist” is used in the broadest senseand includes any molecule that mimics a biological activity of a nativePRO87299 polypeptide disclosed herein. Suitable agonist or antagonistmolecules specifically include agonist or antagonist antibodies orantibody fragments, fragments or amino acid sequence variants of nativePRO87299 polypeptides, peptides, antisense oligonucleotides, smallorganic molecules, etc. Methods for identifying agonists or antagonistsof a PRO87299 polypeptide may comprise contacting a PRO87299 polypeptidewith a candidate agonist or antagonist molecule and measuring adetectable change in one or more biological activities normallyassociated with the PRO87299 polypeptide.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.

“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc. Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An antibody that “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide is onethat binds to that particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label may be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

By “solid phase” is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.,controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a PRO87299 polypeptide or antibody thereto) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

The term “immune related disease” means a disease in which a componentof the immune system of a mammal causes, mediates or otherwisecontributes to a morbidity in the mammal. Also included are diseases inwhich stimulation or intervention of the immune response has anameliorative effect on progression of the disease. Included within thisterm are immune-mediated inflammatory diseases, non-immune-mediatedinflammatory diseases, infectious diseases, immunodeficiency diseases,neoplasia, etc.

The term “T cell mediated disease” means a disease in which T cellsdirectly or indirectly mediate or otherwise contribute to a morbidity ina mammal. The T cell mediated disease may be associated with cellmediated effects, lymphokine mediated effects, etc., and even effectsassociated with B cells if the B cells are stimulated, for example, bythe lymphokines secreted by T cells.

Examples of immune-related and inflammatory diseases, some of which areimmune or T cell mediated, which can be treated according to theinvention include systemic lupus erythematosis, rheumatoid arthritis,juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis(scleroderma), idiopathic inflammatory myopathies (dermatomyositis,polymyositis), Sjögren's syndrome, systemic vasculitis, sarcoidosis,autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria), autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis),demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy, hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis, inflammatory bowel disease(ulcerative colitis: Crohn's disease), gluten-sensitive enteropathy, andWhipple's disease, autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis, allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria, immunologic diseases ofthe lung such as eosinophilic pneumonias, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis, transplantation associated diseasesincluding graft rejection and graft-versus-host-disease. Infectiousdiseases including viral diseases such as AIDS (HIV infection),hepatitis A, B, C, D, and E, herpes, etc., bacterial infections, fungalinfections, protozoal infections and parasitic infections.

The term “effective amount” is a concentration or amount of a PRO87299polypeptide and/or agonist/antagonist which results in achieving aparticular stated purpose. An “effective amount” of a PRO87299polypeptide or agonist or antagonist thereof may be determinedempirically. Furthermore, a “therapeutically effective amount” is aconcentration or amount of a PRO87299 polypeptide and/oragonist/antagonist which is effective for achieving a stated therapeuticeffect. This amount may also be determined empirically.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeadriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosinearabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin,taxoids, e.g., paclitaxel (Taxol, Bristol-Myers Squibb Oncology,Princeton, N.J.), and doxetaxel (Taxotere, Rhône-Poulenc Rorer, Antony,France), toxotere, methotrexate, cisplatin, melphalan, vinblastine,bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone,vincristine, vinorelbine, carboplatin, teniposide, daunomycin,carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins (seeU.S. Pat. No. 4,675,187), melphalan and other related nitrogen mustards.Also included in this definition are hormonal agents that act toregulate or inhibit hormone action on tumors such as tamoxifen andonapristone.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially cancer celloverexpressing any of the genes identified herein, either in vitro or invivo. Thus, the growth inhibitory agent is one which significantlyreduces the percentage of cells overexpressing such genes in S phase.Examples of growth inhibitory agents include agents that block cellcycle progression (at a place other than S phase), such as agents thatinduce G1 arrest and M-phase arrest. Classical M-phase blockers includethe vincas (vincristine and vinblastine), taxol, and topo II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest G1 also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogens, and antineoplastic drugs” by Murakami et al. (WBSaunders: Philadelphia, 1995), especially p. 13.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ,colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α orTNF-β; and other polypeptide factors including LIF and kit ligand (KL).As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

As used herein, the term “inflammatory cells” designates cells thatenhance the inflammatory response such as mononuclear cells,eosinophils, macrophages, and polymorphonuclear neutrophils (PMN).

TABLE 2 PRO87299 XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO87299 polypeptide)= 5 divided by 15 = 33.3%

TABLE 3 PRO87299 XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO87299 polypeptide)= 5 divided by 10 = 50%

TABLE 4 PRO87299- NNNNNNNNNNNNNN (Length = 14 nucleotides) DNAComparison NNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA % nucleic acidsequence identity = (the number of identically matching nucleotidesbetween the two nucleic acid sequences as determined by ALIGN-2) dividedby (the total number of nucleotides of the PRO87299-DNA nucleic acidsequence) = 6 divided by 14 = 42.9%

TABLE 5 PRO87299-DNA NNNNNNNNNNNN (Length = 12 nucleotides) ComparisonDNA NNNNLLLVV (Length = 9 nucleotides) % nucleic acid sequence identity= (the number of identically matching nucleotides between the twonucleic acid sequences as determined by ALIGN-2) divided by (the totalnumber of nucleotides of the PRO87299-DNA nucleic acid sequence) = 4divided by 12 = 33.3%

II. Compositions and Methods of the Invention

A. Full-Length PRO87299 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO87299 polypeptides. In particular, cDNAs encoding various PRO87299polypeptides have been identified and isolated, as disclosed in furtherdetail in the Examples below.

The actual nucleotide sequences of those clones can readily bedetermined by the skilled artisan by sequencing of the deposited cloneusing routine methods in the art. The predicted amino acid sequence canbe determined from the nucleotide sequence using routine skill. For thePRO87299 polypeptides and encoding nucleic acids described herein,Applicants have identified what is believed to be the reading frame bestidentifiable with the sequence information available at the time.

B. PRO87299 Polypeptide Variants

In addition to the full-length native sequence PRO87299 polypeptidesdescribed herein, it is contemplated that PRO87299 variants can beprepared. PRO87299 variants can be prepared by introducing appropriatenucleotide changes into the PRO87299 DNA, and/or by synthesis of thedesired PRO87299 polypeptide. Those skilled in the art will appreciatethat amino acid changes may alter post-translational processes of thePRO87299, such as changing the number or position of glycosylation sitesor altering the membrane anchoring characteristics.

Variations in the native full-length sequence PRO87299 or in variousdomains of the PRO87299 described herein, can be made, for example,using any of the techniques and guidelines for conservative andnon-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion or insertion ofone or more codons encoding the PRO87299 that results in a change in theamino acid sequence of the PRO87299 as compared with the native sequencePRO87299. Optionally, the variation is by substitution of at least oneamino acid with any other amino acid in one or more of the domains ofthe PRO87299. Guidance in determining which amino acid residue may beinserted, substituted or deleted without adversely affecting the desiredactivity may be found by comparing the sequence of the PRO87299 withthat of homologous known protein molecules and minimizing the number ofamino acid sequence changes made in regions of high homology. Amino acidsubstitutions can be the result of replacing one amino acid with anotheramino acid having similar structural and/or chemical properties, such asthe replacement of a leucine with a serine, i.e., conservative aminoacid replacements. Insertions or deletions may optionally be in therange of about 1 to 5 amino acids. The variation allowed may bedetermined by systematically making insertions, deletions orsubstitutions of amino acids in the sequence and testing the resultingvariants for activity exhibited by the full-length or mature nativesequence.

PRO87299 polypeptide fragments are provided herein. Such fragments maybe truncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the PRO87299 polypeptide.

PRO87299 fragments may be prepared by any of a number of conventionaltechniques. Desired peptide fragments may be chemically synthesized. Analternative approach involves generating PRO87299 fragments by enzymaticdigestion, e.g., by treating the protein with an enzyme known to cleaveproteins at sites defined by particular amino acid residues, or bydigesting the DNA with suitable restriction enzymes and isolating thedesired fragment. Yet another suitable technique involves isolating andamplifying a DNA fragment encoding a desired polypeptide fragment, bypolymerase chain reaction (PCR). Oligonucleotides that define thedesired termini of the DNA fragment are employed at the 5′ and 3′primers in the PCR. Preferably, PRO87299 polypeptide fragments share atleast one biological and/or immunological activity with the nativePRO87299 polypeptide disclosed herein.

In particular embodiments, conservative substitutions of interest areshown in Table 6 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 6 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T)ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; leu ala; norleucine

Substantial modifications in function or immunological identity of thePRO87299 polypeptide are accomplished by selecting substitutions thatdiffer significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

-   (1) hydrophobic: norleucine, met, ala, val, leu, ile;-   (2) neutral hydrophilic: cys, ser, thr;-   (3) acidic: asp, glu;-   (4) basic: asn, gln, his, lys, arg;-   (5) residues that influence chain orientation: gly, pro; and-   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO87299 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

C. Modifications of PRO87299

Covalent modifications of PRO87299 are included within the scope of thisinvention. One type of covalent modification includes reacting targetedamino acid residues of a PRO87299 polypeptide with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues of the PRO87299. Derivatization withbifunctional agents is useful, for instance, for crosslinking PRO87299to a water-insoluble support matrix or surface for use in the method forpurifying anti-PRO87299 antibodies, and vice-versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the PRO87299 polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence PRO87299(either by removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequencePRO87299. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present.

Addition of glycosylation sites to the PRO87299 polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence PRO87299 (forO-linked glycosylation sites). The PRO87299 amino acid sequence mayoptionally be altered through changes at the DNA level, particularly bymutating the DNA encoding the PRO87299 polypeptide at preselected basessuch that codons are generated that will translate into the desiredamino acids.

Another means of increasing the number of carbohydrate moieties on thePRO87299 polypeptide is by chemical or enzymatic coupling of glycosidesto the polypeptide. Such methods are described in the art, e.g., in WO87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the PRO87299 polypeptide maybe accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Another type of covalent modification of PRO87299 comprises linking thePRO87299 polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The PRO87299 of the present invention may also be modified in a way toform a chimeric molecule comprising PRO87299 fused to another,heterologous polypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of thePRO87299 with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the PRO87299. The presenceof such epitope-tagged forms of the PRO87299 can be detected using anantibody against the tag polypeptide. Also, provision of the epitope tagenables the PRO87299 to be readily purified by affinity purificationusing an anti-tag antibody or another type of affinity matrix that bindsto the epitope tag. Various tag polypeptides and their respectiveantibodies are well known in the art. Examples include poly-histidine(poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tagpolypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol.,8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 1(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an alpha-tubulin epitope peptide[Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad.Sci. USA, 87:6393-6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of the PRO87299 with an immunoglobulin or a particular region ofan immunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a PRO87299 polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

D. Preparation of PRO87299

The description below relates primarily to production of PRO87299 byculturing cells transformed or transfected with a vector containingPRO87299 nucleic acid. It is, of course, contemplated that alternativemethods, which are well known in the art, may be employed to preparePRO87299. For instance, the PRO87299 sequence, or portions thereof, maybe produced by direct peptide synthesis using solid-phase techniques[see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. FreemanCo., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc.,85:2149-2154 (1963)]. In vitro protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may beaccomplished, for instance, using an Applied Biosystems PeptideSynthesizer (Foster City, Calif.) using manufacturer's instructions.Various portions of the PRO87299 may be chemically synthesizedseparately and combined using chemical or enzymatic methods to producethe full-length PRO87299.

1. Isolation of DNA Encoding PRO87299

DNA encoding PRO87299 may be obtained from a cDNA library prepared fromtissue believed to possess the PRO87299 mRNA and to express it at adetectable level. Accordingly, human PRO87299 DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The PRO87299-encoding gene may also beobtained from a genomic library or by known synthetic procedures (e.g.,automated nucleic acid synthesis).

Libraries can be screened with probes (such as antibodies to thePRO87299 or oligonucleotides of at least about 20-80 bases) designed toidentify the gene of interest or the protein encoded by it. Screeningthe cDNA or genomic library with the selected probe may be conductedusing standard procedures, such as described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989). An alternative means to isolate the geneencoding PRO87299 is to use PCR methodology [Sambrook et al., supra;Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring HarborLaboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for PRO87299 production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvG kan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forPRO87299-encoding vectors. Saccharomyces cerevisiae is a commonly usedlower eukaryotic host microorganism. Others include Schizosaccharomycespombe (Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383 published 2May 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742[1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278[1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene,26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated PRO87299 arederived from multicellular organisms. Examples of invertebrate cellsinclude insect cells such as Drosophila S2 and Spodoptera Sf9, as wellas plant cells. Examples of useful mammalian host cell lines includeChinese hamster ovary (CHO) and COS cells. More specific examplesinclude monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); Chinese hamster ovary cells/−DHFR(CHO, Urlaub and Chasin, Proc.Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather,Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75);human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT060562, ATCC CCL51). The selection of the appropriate host cell isdeemed to be within the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO87299 may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

The PRO87299 may be produced recombinantly not only directly, but alsoas a fusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe PRO87299-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), orthe signal described in WO 90/13646 published 15 Nov. 1990. In mammaliancell expression, mammalian signal sequences may be used to directsecretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up thePRO87299-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the PRO87299-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encodingPRO87299.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

PRO87299 transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g.,the actin promoter or an immunoglobulin promoter, and from heat-shockpromoters, provided such promoters are compatible with the host cellsystems.

Transcription of a DNA encoding the PRO87299 by higher eukaryotes may beincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp, thatact on a promoter to increase its transcription. Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO87299 coding sequence, but is preferably located at a site 5′ fromthe promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding PRO87299.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of PRO87299 in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO87299 polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused toPRO87299 DNA and encoding a specific antibody epitope.

5. Purification of Polypeptide

Forms of PRO87299 may be recovered from culture medium or from host celllysates. If membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of PRO87299 can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

It may be desired to purify PRO87299 from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of thePRO87299. Various methods of protein purification may be employed andsuch methods are known in the art and described for example inDeutscher, Methods in Enzymology, 182 (1990); Scopes, ProteinPurification: Principles and Practice, Springer-Verlag, New York (1982).The purification step(s) selected will depend, for example, on thenature of the production process used and the particular PRO87299produced.

E. Tissue Distribution

The location of tissues expressing the PRO87299 can be identified bydetermining mRNA expression in various human tissues. The location ofsuch genes provides information about which tissues are most likely tobe affected by the stimulating and inhibiting activities of the PRO87299polypeptides. The location of a gene in a specific tissue also providessample tissue for the activity blocking assays discussed below.

As noted before, gene expression in various tissues may be measured byconventional Southern blotting, Northern blotting to quantitate thetranscription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205[1980]), dot blotting (DNA analysis), or in situ hybridization, using anappropriately labeled probe, based on the sequences provided herein.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes.

Gene expression in various tissues, alternatively, may be measured byimmunological methods, such as immunohistochemical staining of tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequenceof a PRO87299 polypeptide or against a synthetic peptide based on theDNA sequences encoding the PRO87299 polypeptide or against an exogenoussequence fused to a DNA encoding a PRO87299 polypeptide and encoding aspecific antibody epitope. General techniques for generating antibodies,and special protocols for Northern blotting and in situ hybridizationare provided below.

F. Antibody Binding Studies

The activity of the PRO87299 polypeptides can be further verified byantibody binding studies, in which the ability of anti-PRO87299antibodies to inhibit the effect of the PRO87299 polypeptides,respectively, on tissue cells is tested. Exemplary antibodies includepolyclonal, monoclonal, humanized, bispecific, and heteroconjugateantibodies, the preparation of which will be described hereinbelow.

Antibody binding studies may be carried out in any known assay method,such as competitive binding assays, direct and indirect sandwich assays,and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual ofTechniques, pp. 147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of target protein in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies preferably are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

For immunohistochemistry, the tissue sample may be fresh or frozen ormay be embedded in paraffin and fixed with a preservative such asformalin, for example.

G. Cell-Based Assays

Cell-based assays and animal models for immune related diseases can beused to further understand the relationship between the genes andpolypeptides identified herein and the development and pathogenesis ofimmune related disease.

In a different approach, cells of a cell type known to be involved in aparticular immune related disease are transfected with the cDNAsdescribed herein, and the ability of these cDNAs to stimulate or inhibitimmune function is analyzed. Suitable cells can be transfected with thedesired gene, and monitored for immune function activity. Suchtransfected cell lines can then be used to test the ability of poly- ormonoclonal antibodies or antibody compositions to inhibit or stimulateimmune function, for example to modulate T-cell proliferation orinflammatory cell infiltration. Cells transfected with the codingsequences of the genes identified herein can further be used to identifydrug candidates for the treatment of immune related diseases.

In addition, primary cultures derived from transgenic animals (asdescribed below) can be used in the cell-based assays herein, althoughstable cell lines are preferred. Techniques to derive continuous celllines from transgenic animals are well known in the art (see, e.g.,Small et al., Mol. Cell. Biol. 5: 642-648 [1985]).

One suitable cell based assay is the mixed lymphocyte reaction (MLR).Current Protocols in Immunology, unit 3.12; edited by J E Coligan, A MKruisbeek, D H Marglies, E M Shevach, W Strober, National Institutes ofHealth, Published by John Wiley & Sons, Inc. In this assay, the abilityof a test compound to stimulate or inhibit the proliferation ofactivated T cells is assayed. A suspension of responder T cells iscultured with allogeneic stimulator cells and the proliferation of Tcells is measured by uptake of tritiated thymidine. This assay is ageneral measure of T cell reactivity. Since the majority of T cellsrespond to and produce IL-2 upon activation, differences inresponsiveness in this assay in part reflect differences in IL-2production by the responding cells. The MLR results can be verified by astandard lymphokine (IL-2) detection assay. Current Protocols inImmunology, above, 3.15, 6.3.

A proliferative T cell response in an MLR assay may be due to directmitogenic properties of an assayed molecule or to external antigeninduced activation. T cell activation requires an antigen specificsignal mediated through the T-cell receptor (TCR) and a costimulatorysignal mediated through a second ligand binding interaction, forexample, the B7 (CD80, CD86)/CD28 binding interaction. CD28 crosslinkingincreases lymphokine secretion by activated T cells. T cell activationhas both negative and positive controls through the binding of ligandswhich have a negative or positive effect. CD28 and CTLA-4 are relatedglycoproteins in the Ig superfamily which bind to B7. CD28 binding to B7has a positive costimulation effect of T cell activation; conversely,CTLA-4 binding to B7 has a T cell deactivating effect. Chambers, C. A.and Allison, J. P., Curr. Opin. Immunol. (1997) 9:396. Schwartz, R. H.,Cell (1992) 71:1065; Linsey, P. S. and Ledbetter, J. A., Annu. Rev.Immunol. (1993) 11:191; June, C. H. et al, Immunol. Today (1994) 15:321;Jenkins, M. K., Immunity (1994) 1:405. In a costimulation assay, thePRO87299 polypeptides are assayed for T cell costimulatory or inhibitoryactivity.

Direct use of a stimulating compound as in the invention has beenvalidated in experiments with 4-1BB glycoprotein, a member of the tumornecrosis factor receptor family, which binds to a ligand (4-1BBL)expressed on primed T cells and signals T cell activation and growth.Alderson, M. E. et al., J. Immunol. (1994) 24:2219.

The use of an agonist stimulating compound has also been validatedexperimentally. Activation of 4-1BB by treatment with an agonistanti-4-1BB antibody enhances eradication of tumors. Hellstrom, I. andHellstrom, K. E., Crit. Rev. Immunol. (1998) 18:1. Immunoadjuvanttherapy for treatment of tumors, described in more detail below, isanother example of the use of the stimulating compounds of theinvention.

Alternatively, an immune stimulating or enhancing effect can also beachieved by administration of a PRO87299 which has vascular permeabilityenhancing properties. Enhanced vascular permeability would be beneficialto disorders which can be attenuated by local infiltration of immunecells (e.g., monocytes, eosinophils, PMNs) and inflammation.

On the other hand, PRO87299 polypeptides, as well as other compounds ofthe invention, which are direct inhibitors of T cellproliferation/activation, lymphokine secretion, and/or vascularpermeability can be directly used to suppress the immune response. Thesecompounds are useful to reduce the degree of the immune response and totreat immune related diseases characterized by a hyperactive,superoptimal, or autoimmune response. This use of the compounds of theinvention has been validated by the experiments described above in whichCTLA-4 binding to receptor B7 deactivates T cells. The direct inhibitorycompounds of the invention function in an analogous manner. The use ofcompound which suppress vascular permeability would be expected toreduce inflammation. Such uses would be beneficial in treatingconditions associated with excessive inflammation.

Alternatively, compounds, e.g., antibodies, which bind to stimulatingPRO87299 polypeptides and block the stimulating effect of thesemolecules produce a net inhibitory effect and can be used to suppressthe T cell mediated immune response by inhibiting T cellproliferation/activation and/or lymphokine secretion. Blocking thestimulating effect of the polypeptides suppresses the immune response ofthe mammal. This use has been validated in experiments using an anti-IL2antibody. In these experiments, the antibody binds to IL2 and blocksbinding of IL2 to its receptor thereby achieving a T cell inhibitoryeffect.

H. Animal Models

The results of the cell based in vitro assays can be further verifiedusing in vivo animal models and assays for T-cell function. A variety ofwell known animal models can be used to further understand the role ofthe genes identified herein in the development and pathogenesis ofimmune related disease, and to test the efficacy of candidatetherapeutic agents, including antibodies, and other antagonists of thenative polypeptides, including small molecule antagonists. The in vivonature of such models makes them predictive of responses in humanpatients. Animal models of immune related diseases include bothnon-recombinant and recombinant (transgenic) animals. Non-recombinantanimal models include, for example, rodent, e.g., murine models. Suchmodels can be generated by introducing cells into syngeneic mice usingstandard techniques, e.g., subcutaneous injection, tail vein injection,spleen implantation, intraperitoneal implantation, implantation underthe renal capsule, etc.

Graft-versus-host disease occurs when immunocompetent cells aretransplanted into immunosuppressed or tolerant patients. The donor cellsrecognize and respond to host antigens. The response can vary from lifethreatening severe inflammation to mild cases of diarrhea and weightloss. Graft-versus-host disease models provide a means of assessing Tcell reactivity against MHC antigens and minor transplant antigens. Asuitable procedure is described in detail in Current Protocols inImmunology, above, unit 4.3.

An animal model for skin allograft rejection is a means of testing theability of T cells to mediate in vivo tissue destruction and a measureof their role in transplant rejection. The most common and acceptedmodels use murine tail-skin grafts. Repeated experiments have shown thatskin allograft rejection is mediated by T cells, helper T cells andkiller-effector T cells, and not antibodies. Auchincloss, H. Jr. andSachs, D. H., Fundamental Immunology, 2nd ed., W. E. Paul ed., RavenPress, NY, 1989, 889-992. A suitable procedure is described in detail inCurrent Protocols in Immunology, above, unit 4.4. Other transplantrejection models which can be used to test the compounds of theinvention are the allogeneic heart transplant models described byTanabe, M. et al, Transplantation (1994) 58:23 and Tinubu, S. A. et al,J. Immunol. (1994) 4330-4338.

Animal models for delayed type hypersensitivity provides an assay ofcell mediated immune function as well. Delayed type hypersensitivityreactions are a T cell mediated in vivo immune response characterized byinflammation which does not reach a peak until after a period of timehas elapsed after challenge with an antigen. These reactions also occurin tissue specific autoimmune diseases such as multiple sclerosis (MS)and experimental autoimmune encephalomyelitis (EAE, a model for MS). Asuitable procedure is described in detail in Current Protocols inImmunology, above, unit 4.5.

EAE is a T cell mediated autoimmune disease characterized by T cell andmononuclear cell inflammation and subsequent demyelination of axons inthe central nervous system. EAE is generally considered to be a relevantanimal model for MS in humans. Bolton, C., Multiple Sclerosis (1995)1:143. Both acute and relapsing-remitting models have been developed.The compounds of the invention can be tested for T cell stimulatory orinhibitory activity against immune mediated demyelinating disease usingthe protocol described in Current Protocols in Immunology, above, units15.1 and 15.2. See also the models for myelin disease in whicholigodendrocytes or Schwann cells are grafted into the central nervoussystem as described in Duncan, I. D. et al, Molec. Med. Today (1997)554-561.

Contact hypersensitivity is a simple delayed type hypersensitivity invivo assay of cell mediated immune function. In this procedure,cutaneous exposure to exogenous haptens which gives rise to a delayedtype hypersensitivity reaction which is measured and quantitated.Contact sensitivity involves an initial sensitizing phase followed by anelicitation phase. The elicitation phase occurs when the T lymphocytesencounter an antigen to which they have had previous contact. Swellingand inflammation occur, making this an excellent model of human allergiccontact dermatitis. A suitable procedure is described in detail inCurrent Protocols in Immunology, Eds. J. E. Cologan, A. M. Kruisbeek, D.H. Margulies, E. M. Shevach and W. Strober, John Wiley & Sons, Inc.,1994, unit 4.2. See also Grabbe, S. and Schwarz, T, Immun. Today 19 (1):37-44 (1998).

An animal model for arthritis is collagen-induced arthritis. This modelshares clinical, histological and immunological characteristics of humanautoimmune rheumatoid arthritis and is an acceptable model for humanautoimmune arthritis. Mouse and rat models are characterized bysynovitis, erosion of cartilage and subchondral bone. The compounds ofthe invention can be tested for activity against autoimmune arthritisusing the protocols described in Current Protocols in Immunology, above,units 15.5. See also the model using a monoclonal antibody to CD18 andVLA-4 integrins described in Issekutz, A. C. et al., Immunology (1996)88:569.

A model of asthma has been described in which antigen-induced airwayhyper-reactivity, pulmonary eosinophilia and inflammation are induced bysensitizing an animal with ovalbumin and then challenging the animalwith the same protein delivered by aerosol. Several animal models(guinea pig, rat, non-human primate) show symptoms similar to atopicasthma in humans upon challenge with aerosol antigens. Murine modelshave many of the features of human asthma. Suitable procedures to testthe compounds of the invention for activity and effectiveness in thetreatment of asthma are described by Wolyniec, W. W. et al, Am. J.Respir. Cell Mol. Biol. (1998) 18:777 and the references cited therein.

Additionally, the compounds of the invention can be tested on animalmodels for psoriasis like diseases. Evidence suggests a T cellpathogenesis for psoriasis. The compounds of the invention can be testedin the scid/scid mouse model described by Schon, M. P. et al, Nat. Med.(1997) 3:183, in which the mice demonstrate histopathologic skin lesionsresembling psoriasis. Another suitable model is the human skin/scidmouse chimera prepared as described by Nickoloff, B. J. et al, Am. J.Path. (1995) 146:580.

Recombinant (transgenic) animal models can be engineered by introducingthe coding portion of the genes identified herein into the genome ofanimals of interest, using standard techniques for producing transgenicanimals. Animals that can serve as a target for transgenic manipulationinclude, without limitation, mice, rats, rabbits, guinea pigs, sheep,goats, pigs, and non-human primates, e.g., baboons, chimpanzees andmonkeys. Techniques known in the art to introduce a transgene into suchanimals include pronucleic microinjection (Hoppe and Wanger, U.S. Pat.No. 4,873,191); retrovirus-mediated gene transfer into germ lines (e.g.,Van der Putten et al., Proc. Natl. Acad. Sci. USA 82 6148-615 [1985]);gene targeting in embryonic stem cells (Thompson et al., Cell 56,313-321 [1989]); electroporation of embryos (Lo, Mol. Cel. Biol. 3,1803-1814 [1983]); sperm-mediated gene transfer (Lavitrano et al., Cell57 717-73 [1989]). For review, see, for example, U.S. Pat. No.4,736,866.

For the purpose of the present invention, transgenic animals includethose that carry the transgene only in part of their cells (“mosaicanimals”). The transgene can be integrated either as a single transgene,or in concatamers, e.g., head-to-head or head-to-tail tandems. Selectiveintroduction of a transgene into a particular cell type is also possibleby following, for example, the technique of Lasko et al., Proc. Natl.Acad. Sci. USA 89 6232-636 (1992).

The expression of the transgene in transgenic animals can be monitoredby standard techniques. For example, Southern blot analysis or PCRamplification can be used to verify the integration of the transgene.The level of mRNA expression can then be analyzed using techniques suchas in situ hybridization, Northern blot analysis, PCR, orimmunocytochemistry.

The animals may be further examined for signs of immune diseasepathology, for example by histological examination to determineinfiltration of immune cells into specific tissues. Blocking experimentscan also be performed in which the transgenic animals are treated withthe compounds of the invention to determine the extent of the T cellproliferation stimulation or inhibition of the compounds. In theseexperiments, blocking antibodies which bind to the PRO87299 polypeptide,prepared as described above, are administered to the animal and theeffect on immune function is determined.

Alternatively, “knock out” animals can be constructed which have adefective or altered gene encoding a polypeptide identified herein, as aresult of homologous recombination between the endogenous gene encodingthe polypeptide and altered genomic DNA encoding the same polypeptideintroduced into an embryonic cell of the animal. For example, cDNAencoding a particular polypeptide can be used to clone genomic DNAencoding that polypeptide in accordance with established techniques. Aportion of the genomic DNA encoding a particular polypeptide can bedeleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected [see e.g., Li et al.,Cell, 69:915 (1992)]. The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-152]. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the polypeptide.

I. ImmunoAdjuvant Therapy

In one embodiment, the immunostimulating compounds of the invention canbe used in immunoadjuvant therapy for the treatment of tumors (cancer).It is now well established that T cells recognize human tumor specificantigens. One group of tumor antigens, encoded by the MAGE, BAGE andGAGE families of genes, are silent in all adult normal tissues, but areexpressed in significant amounts in tumors, such as melanomas, lungtumors, head and neck tumors, and bladder carcinomas. DeSmet, C. et al.,(1996) Proc. Natl. Acad. Sci. USA, 93:7149. It has been shown thatcostimulation of T cells induces tumor regression and an antitumorresponse both in vitro and in vivo. Melero, I. et al., Nature Medicine(1997) 3:682; Kwon, E. D. et al., Proc. Natl. Acad. Sci. USA (1997) 94:8099; Lynch, D. H. et al, Nature Medicine (1997) 3:625; Finn, O. J. andLotze, M. T., J. Immunol. (1998) 21:114. The stimulatory compounds ofthe invention can be administered as adjuvants, alone or together with agrowth regulating agent, cytotoxic agent or chemotherapeutic agent, tostimulate T cell proliferation/activation and an antitumor response totumor antigens. The growth regulating, cytotoxic, or chemotherapeuticagent may be administered in conventional amounts using knownadministration regimes. Immunostimulating activity by the compounds ofthe invention allows reduced amounts of the growth regulating,cytotoxic, or chemotherapeutic agents thereby potentially lowering thetoxicity to the patient.

J. Screening Assays for Drug Candidates

Screening assays for drug candidates are designed to identify compoundsthat bind to or complex with the polypeptides encoded by the genesidentified herein or a biologically active fragment thereof, orotherwise interfere with the interaction of the encoded polypeptideswith other cellular proteins. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.Small molecules contemplated include synthetic organic or inorganiccompounds, including peptides, preferably soluble peptides,(poly)peptide-immunoglobulin fusions, and, in particular, antibodiesincluding, without limitation, poly- and monoclonal antibodies andantibody fragments, single-chain antibodies, anti-idiotypic antibodies,and chimeric or humanized versions of such antibodies or fragments, aswell as human antibodies and antibody fragments. The assays can beperformed in a variety of formats, including protein-protein bindingassays, biochemical screening assays, immunoassays and cell basedassays, which are well characterized in the art. All assays are commonin that they call for contacting the drug candidate with a polypeptideencoded by a nucleic acid identified herein under conditions and for atime sufficient to allow these two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the polypeptide encoded by the gene identified herein or thedrug candidate is immobilized on a solid phase, e.g., on a microtiterplate, by covalent or non-covalent attachments. Non-covalent attachmentgenerally is accomplished by coating the solid surface with a solutionof the polypeptide and drying. Alternatively, an immobilized antibody,e.g., a monoclonal antibody, specific for the polypeptide to beimmobilized can be used to anchor it to a solid surface. The assay isperformed by adding the non-immobilized component, which may be labeledby a detectable label, to the immobilized component, e.g., the coatedsurface containing the anchored component. When the reaction iscomplete, the non-reacted components are removed, e.g., by washing, andcomplexes anchored on the solid surface are detected. When theoriginally non-immobilized component carries a detectable label, thedetection of label immobilized on the surface indicates that complexingoccurred. Where the originally non-immobilized component does not carrya label, complexing can be detected, for example, by using a labelledantibody specifically binding the immobilized complex.

If the candidate compound interacts with but does not bind to aparticular protein encoded by a gene identified herein, its interactionwith that protein can be assayed by methods well known for detectingprotein-protein interactions. Such assays include traditionalapproaches, such as, cross-linking, co-immunoprecipitation, andco-purification through gradients or chromatographic columns. Inaddition, protein-protein interactions can be monitored by using ayeast-based genetic system described by Fields and co-workers [Fieldsand Song, Nature (London) 340, 245-246 (1989); Chien et al., Proc. Natl.Acad. Sci. USA 88, 9578-9582 (1991)] as disclosed by Chevray andNathans, Proc. Natl. Acad. Sci. USA 89 5789-5793 (1991). Manytranscriptional activators, such as yeast GAL4, consist of twophysically discrete modular domains, one acting as the DNA-bindingdomain, while the other one functioning as the transcription activationdomain. The yeast expression system described in the foregoingpublications (generally referred to as the “two-hybrid system”) takesadvantage of this property, and employs two hybrid proteins, one inwhich the target protein is fused to the DNA-binding domain of GALA, andanother, in which candidate activating proteins are fused to theactivation domain. The expression of a GAL1-lacZ reporter gene undercontrol of a GAL4-activated promoter depends on reconstitution of GALAactivity via protein-protein interaction. Colonies containinginteracting polypeptides are detected with a chromogenic substrate forβ-galactosidase. A complete kit (MATCHMAKER™) for identifyingprotein-protein interactions between two specific proteins using thetwo-hybrid technique is commercially available from Clontech. Thissystem can also be extended to map protein domains involved in specificprotein interactions as well as to pinpoint amino acid residues that arecrucial for these interactions.

In order to find compounds that interfere with the interaction of a geneidentified herein and other intra- or extracellular components can betested, a reaction mixture is usually prepared containing the product ofthe gene and the intra- or extracellular component under conditions andfor a time allowing for the interaction and binding of the two products.To test the ability of a test compound to inhibit binding, the reactionis run in the absence and in the presence of the test compound. Inaddition, a placebo may be added to a third reaction mixture, to serveas positive control. The binding (complex formation) between the testcompound and the intra- or extracellular component present in themixture is monitored as described above. The formation of a complex inthe control reaction(s) but not in the reaction mixture containing thetest compound indicates that the test compound interferes with theinteraction of the test compound and its reaction partner.

K. Compositions and Methods for the Treatment of Immune Related Diseases

The compositions useful in the treatment of immune related diseasesinclude, without limitation, proteins, antibodies, small organicmolecules, peptides, phosphopeptides, antisense and ribozyme molecules,triple helix molecules, etc. that inhibit or stimulate immune function,for example, T cell proliferation/activation, lymphokine release, orimmune cell infiltration.

For example, antisense RNA and RNA molecules act to directly block thetranslation of mRNA by hybridizing to targeted mRNA and preventingprotein translation. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation initiation site,e.g., between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology 4, 469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple helix formation via Hoogsteen basepairing rules, which generally require sizeable stretches of purines orpyrimidines on one strand of a duplex. For further details see, e.g.,PCT publication No. WO 97/33551, supra.

These molecules can be identified by any or any combination of thescreening assays discussed above and/or by any other screeningtechniques well known for those skilled in the art.

L. Anti-PRO87299 Antibodies

The present invention further provides anti-PRO87299 antibodies.Exemplary antibodies include polyclonal, monoclonal, humanized,bispecific, and heteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-PRO87299 antibodies may comprise polyclonal antibodies. Methodsof preparing polyclonal antibodies are known to the skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the PRO87299 polypeptide or a fusionprotein thereof. It may be useful to conjugate the immunizing agent to aprotein known to be immunogenic in the mammal being immunized. Examplesof such immunogenic proteins include but are not limited to keyholelimpet hemocyanin, serum albumin, bovine thyroglobulin, and soybeantrypsin inhibitor. Examples of adjuvants which may be employed includeFreund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

The anti-PRO87299 antibodies may, alternatively, be monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The immunizing agent will typically include the PRO87299 polypeptide ora fusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed againstPRO87299. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

3. Human and Humanized Antibodies

The anti-PRO87299 antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

The antibodies may also be affinity matured using known selection and/ormutagenesis methods as described above. Preferred affinity maturedantibodies have an affinity which is five times, more preferably 10times, even more preferably 20 or 30 times greater than the startingantibody (generally murine, humanized or human) from which the maturedantibody is prepared.

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe PRO87299, the other one is for any other antigen, and preferably fora cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., Science229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med.175:217-225 (1992) describe the production of a fully humanizedbispecific antibody F(ab′)₂ molecule. Each Fab′ fragment was separatelysecreted from E. coli and subjected to directed chemical coupling invitro to form the bispecific antibody. The bispecific antibody thusformed was able to bind to cells overexpressing the ErbB2 receptor andnormal human T cells, as well as trigger the lytic activity of humancytotoxic lymphocytes against human breast tumor targets.

Various technique for making and isolating bispecific antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bispecific antibodies have been produced using leucine zippers.Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipperpeptides from the Fos and Jun proteins were linked to the Fab′ portionsof two different antibodies by gene fusion. The antibody homodimers werereduced at the hinge region to form monomers and then re-oxidized toform the antibody heterodimers. This method can also be utilized for theproduction of antibody homodimers. The “diabody” technology described byHollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) hasprovided an alternative mechanism for making bispecific antibodyfragments. The fragments comprise a heavy-chain variable domain (V_(H))connected to a light-chain variable domain (V_(L)) by a linker which istoo short to allow pairing between the two domains on the same chain.Accordingly, the V_(H) and V_(L) domains of one fragment are forced topair with the complementary V_(L) and V_(H) domains of another fragment,thereby forming two antigen-binding sites. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (sFv) dimershas also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies may bind to two different epitopes on agiven PRO87299 polypeptide herein. Alternatively, an anti-PRO87299polypeptide arm may be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2,CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64),FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defensemechanisms to the cell expressing the particular PRO87299 polypeptide.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express a particular PRO87299 polypeptide. These antibodiespossess a PRO87299-binding arm and an arm which binds a cytotoxic agentor a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Anotherbispecific antibody of interest binds the PRO87299 polypeptide andfurther binds tissue factor (TF).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

6. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) may beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al, J. Exp Med., 176: 1191-1195 (1992)and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodieswith enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

7. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is conjugatedto a cytotoxic agent (e.g., a radionucleotide).

8. Immunoliposomes

The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.,81(19): 1484 (1989).

M. Pharmaceutical Compositions

The active PRO87299 molecules of the invention (e.g., PRO87299polypeptides, anti-PRO87299 antibodies, and/or variants of each) as wellas other molecules identified by the screening assays disclosed above,can be administered for the treatment of immune related diseases, in theform of pharmaceutical compositions.

Therapeutic formulations of the active PRO87299 molecule, preferably apolypeptide or antibody of the invention, are prepared for storage bymixing the active molecule having the desired degree of purity withoptional pharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Compounds identified by the screening assays disclosed herein can beformulated in an analogous manner, using standard techniques well knownin the art.

Lipofections or liposomes can also be used to deliver the PRO87299molecule into cells. Where antibody fragments are used, the smallestinhibitory fragment which specifically binds to the binding domain ofthe target protein is preferred. For example, based upon the variableregion sequences of an antibody, peptide molecules can be designed whichretain the ability to bind the target protein sequence. Such peptidescan be synthesized chemically and/or produced by recombinant DNAtechnology (see, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA 90,7889-7893 [1993]).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition may comprise a cytotoxicagent, cytokine or growth inhibitory agent. Such molecules are suitablypresent in combination in amounts that are effective for the purposeintended.

The active PRO87299 molecules may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations or the PRO87299 molecules may beprepared. Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theantibody, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated antibodies remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

N. Methods of Treatment

It is contemplated that the polypeptides, antibodies and other activecompounds of the present invention may be used to treat various immunerelated diseases and conditions, such as T cell mediated diseases,including those characterized by infiltration of inflammatory cells intoa tissue, stimulation of T-cell proliferation, inhibition of T-cellproliferation, increased or decreased vascular permeability or theinhibition thereof.

Exemplary conditions or disorders to be treated with the polypeptides,antibodies and other compounds of the invention, include, but are notlimited to systemic lupus erythematosis, rheumatoid arthritis, juvenilechronic arthritis, osteoarthritis, spondyloarthropathies, systemicsclerosis (scleroderma), idiopathic inflammatory myopathies(dermatomyositis, polymyositis), Sjögren's syndrome, systemicvasculitis, sarcoidosis, autoimmune hemolytic anemia (immunepancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia), thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis),diabetes mellitus, immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis), demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barré syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory bowel disease (ulcerative colitis: Crohn's disease),gluten-sensitive enteropathy, and Whipple's disease, autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis, allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonias, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, transplantation associated diseases including graftrejection and graft-versus-host-disease.

In systemic lupus erythematosus, the central mediator of disease is theproduction of auto-reactive antibodies to self proteins/tissues and thesubsequent generation of immune-mediated inflammation. Antibodies eitherdirectly or indirectly mediate tissue injury. Though T lymphocytes havenot been shown to be directly involved in tissue damage, T lymphocytesare required for the development of auto-reactive antibodies. Thegenesis of the disease is thus T lymphocyte dependent. Multiple organsand systems are affected clinically including kidney, lung,musculoskeletal system, mucocutaneous, eye, central nervous system,cardiovascular system, gastrointestinal tract, bone marrow and blood.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatorydisease that mainly involves the synovial membrane of multiple jointswith resultant injury to the articular cartilage. The pathogenesis is Tlymphocyte dependent and is associated with the production of rheumatoidfactors, auto-antibodies directed against self IgG, with the resultantformation of immune complexes that attain high levels in joint fluid andblood. These complexes in the joint may induce the marked infiltrate oflymphocytes and monocytes into the synovium and subsequent markedsynovial changes; the joint space/fluid if infiltrated by similar cellswith the addition of numerous neutrophils. Tissues affected areprimarily the joints, often in symmetrical pattern. However,extra-articular disease also occurs in two major forms. One form is thedevelopment of extra-articular lesions with ongoing progressive jointdisease and typical lesions of pulmonary fibrosis, vasculitis, andcutaneous ulcers. The second form of extra-articular disease is the socalled Felty's syndrome which occurs late in the RA disease course,sometimes after joint disease has become quiescent, and involves thepresence of neutropenia, thrombocytopenia and splenomegaly. This can beaccompanied by vasculitis in multiple organs with formations ofinfarcts, skin ulcers and gangrene. Patients often also developrheumatoid nodules in the subcutis tissue overlying affected joints; thenodules late stage have necrotic centers surrounded by a mixedinflammatory cell infiltrate. Other manifestations which can occur in RAinclude: pericarditis, pleuritis, coronary arteritis, intestitialpneumonitis with pulmonary fibrosis, keratoconjunctivitis sicca, andrhematoid nodules.

Juvenile chronic arthritis is a chronic idiopathic inflammatory diseasewhich begins often at less than 16 years of age. Its phenotype has somesimilarities to RA; some patients which are rhematoid factor positiveare classified as juvenile rheumatoid arthritis. The disease issub-classified into three major categories: pauciarticular,polyarticular, and systemic. The arthritis can be severe and istypically destructive and leads to joint ankylosis and retarded growth.Other manifestations can include chronic anterior uveitis and systemicamyloidosis.

Spondyloarthropathies are a group of disorders with some common clinicalfeatures and the common association with the expression of HLA-B27 geneproduct. The disorders include: ankylosing sponylitis, Reiter's syndrome(reactive arthritis), arthritis associated with inflammatory boweldisease, spondylitis associated with psoriasis, juvenile onsetspondyloarthropathy and undifferentiated spondyloarthropathy.Distinguishing features include sacroileitis with or withoutspondylitis; inflammatory asymmetric arthritis; association with HLA-B27(a serologically defined allele of the HLA-B locus of class I MHC);ocular inflammation, and absence of autoantibodies associated with otherrheumatoid disease. The cell most implicated as key to induction of thedisease is the CD8+ T lymphocyte, a cell which targets antigen presentedby class I MHC molecules. CD8+ T cells may react against the class I MHCallele HLA-B27 as if it were a foreign peptide expressed by MHC class Imolecules. It has been hypothesized that an epitope of HLA-B27 may mimica bacterial or other microbial antigenic epitope and thus induce a CD8+T cells response.

Systemic sclerosis (scleroderma) has an unknown etiology. A hallmark ofthe disease is induration of the skin; likely this is induced by anactive inflammatory process. Scleroderma can be localized or systemic;vascular lesions are common and endothelial cell injury in themicrovasculature is an early and important event in the development ofsystemic sclerosis; the vascular injury may be immune mediated. Animmunologic basis is implied by the presence of mononuclear cellinfiltrates in the cutaneous lesions and the presence of anti-nuclearantibodies in many patients. ICAM-1 is often upregulated on the cellsurface of fibroblasts in skin lesions suggesting that T cellinteraction with these cells may have a role in the pathogenesis of thedisease. Other organs involved include: the gastrointestinal tract:smooth muscle atrophy and fibrosis resulting in abnormalperistalsis/motility; kidney: concentric subendothelial intimalproliferation affecting small arcuate and interlobular arteries withresultant reduced renal cortical blood flow, results in proteinuria,azotemia and hypertension; skeletal muscle: atrophy, interstitialfibrosis; inflammation; lung: interstitial pneumonitis and interstitialfibrosis; and heart: contraction band necrosis, scarring/fibrosis.

Idiopathic inflammatory myopathies including dermatomyositis,polymyositis and others are disorders of chronic muscle inflammation ofunknown etiology resulting in muscle weakness. Muscleinjury/inflammation is often symmetric and progressive. Autoantibodiesare associated with most forms. These myositis-specific autoantibodiesare directed against and inhibit the function of components, proteinsand RNA's, involved in protein synthesis.

Sjögren's syndrome is due to immune-mediated inflammation and subsequentfunctional destruction of the tear glands and salivary glands. Thedisease can be associated with or accompanied by inflammatory connectivetissue diseases. The disease is associated with autoantibody productionagainst Ro and La antigens, both of which are small RNA-proteincomplexes. Lesions result in keratoconjunctivitis sicca, xerostomia,with other manifestations or associations including bilary cirrhosis,peripheral or sensory neuropathy, and palpable purpura.

Systemic vasculitis are diseases in which the primary lesion isinflammation and subsequent damage to blood vessels which results inischemia/necrosis/degeneration to tissues supplied by the affectedvessels and eventual end-organ dysfunction in some cases. Vasculitidescan also occur as a secondary lesion or sequelae to otherimmune-inflammatory mediated diseases such as rheumatoid arthritis,systemic sclerosis, etc., particularly in diseases also associated withthe formation of immune complexes. Diseases in the primary systemicvasculitis group include: systemic necrotizing vasculitis: polyarteritisnodosa, allergic angiitis and granulomatosis, polyangiitis; Wegener'sgranulomatosis; lymphomatoid granulomatosis; and giant cell arteritis.Miscellaneous vasculitides include: mucocutaneous lymph node syndrome(MLNS or Kawasaki's disease), isolated CNS vasculitis, Behet's disease,thromboangiitis obliterans (Buerger's disease) and cutaneous necrotizingvenulitis. The pathogenic mechanism of most of the types of vasculitislisted is believed to be primarily due to the deposition ofimmunoglobulin complexes in the vessel wall and subsequent induction ofan inflammatory response either via ADCC, complement activation, orboth.

Sarcoidosis is a condition of unknown etiology which is characterized bythe presence of epithelioid granulomas in nearly any tissue in the body;involvement of the lung is most common. The pathogenesis involves thepersistence of activated macrophages and lymphoid cells at sites of thedisease with subsequent chronic sequelae resultant from the release oflocally and systemically active products released by these cell types.

Autoimmune hemolytic anemia including autoimmune hemolytic anemia,immune pancytopenia, and paroxysmal noctural hemoglobinuria is a resultof production of antibodies that react with antigens expressed on thesurface of red blood cells (and in some cases other blood cellsincluding platelets as well) and is a reflection of the removal of thoseantibody coated cells via complement mediated lysis and/orADCC/Fc-receptor-mediated mechanisms.

In autoimmune thrombocytopenia including thrombocytopenic purpura, andimmune-mediated thrombocytopenia in other clinical settings, plateletdestruction/removal occurs as a result of either antibody or complementattaching to platelets and subsequent removal by complement lysis, ADCCor FC-receptor mediated mechanisms.

Thyroiditis including Grave's disease, Hashimoto's thyroiditis, juvenilelymphocytic thyroiditis, and atrophic thyroiditis, are the result of anautoimmune response against thyroid antigens with production ofantibodies that react with proteins present in and often specific forthe thyroid gland. Experimental models exist including spontaneousmodels: rats (BUF and BB rats) and chickens (obese chicken strain);inducible models: immunization of animals with either thyroglobulin,thyroid microsomal antigen (thyroid peroxidase).

Type I diabetes mellitus or insulin-dependent diabetes is the autoimmunedestruction of pancreatic islet β cells; this destruction is mediated byauto-antibodies and auto-reactive T cells. Antibodies to insulin or theinsulin receptor can also produce the phenotype ofinsulin-non-responsiveness.

Immune mediated renal diseases, including glomerulonephritis andtubulointerstitial nephritis, are the result of antibody or T lymphocytemediated injury to renal tissue either directly as a result of theproduction of autoreactive antibodies or T cells against renal antigensor indirectly as a result of the deposition of antibodies and/or immunecomplexes in the kidney that are reactive against other, non-renalantigens. Thus other immune-mediated diseases that result in theformation of immune-complexes can also induce immune mediated renaldisease as an indirect sequelae. Both direct and indirect immunemechanisms result in inflammatory response that produces/induces lesiondevelopment in renal tissues with resultant organ function impairmentand in some cases progression to renal failure. Both humoral andcellular immune mechanisms can be involved in the pathogenesis oflesions.

Demyelinating diseases of the central and peripheral nervous systems,including Multiple Sclerosis; idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome; and Chronic Inflammatory DemyelinatingPolyneuropathy, are believed to have an autoimmune basis and result innerve demyelination as a result of damage caused to oligodendrocytes orto myelin directly. In MS there is evidence to suggest that diseaseinduction and progression is dependent on T lymphocytes. MultipleSclerosis is a demyelinating disease that is T lymphocyte-dependent andhas either a relapsing-remitting course or a chronic progressive course.The etiology is unknown; however, viral infections, geneticpredisposition, environment, and autoimmunity all contribute. Lesionscontain infiltrates of predominantly T lymphocyte mediated, microglialcells and infiltrating macrophages; CD4+ T lymphocytes are thepredominant cell type at lesions. The mechanism of oligodendrocyte celldeath and subsequent demyelination is not known but is likely Tlymphocyte driven.

Inflammatory and Fibrotic Lung Disease, including EosinophilicPneumonias; Idiopathic Pulmonary Fibrosis, and HypersensitivityPneumonitis may involve a disregulated immune-inflammatory response.Inhibition of that response would be of therapeutic benefit.

Autoimmune or Immune-mediated Skin Disease including Bullous SkinDiseases, Erythema Multiforme, and Contact Dermatitis are mediated byauto-antibodies, the genesis of which is T lymphocyte-dependent.

Psoriasis is a T lymphocyte-mediated inflammatory disease. Lesionscontain infiltrates of T lymphocytes, macrophages and antigen processingcells, and some neutrophils.

Allergic diseases, including asthma; allergic rhinitis; atopicdermatitis; food hypersensitivity; and urticaria are T lymphocytedependent. These diseases are predominantly mediated by T lymphocyteinduced inflammation, IgE mediated-inflammation or a combination ofboth.

Transplantation associated diseases, including Graft rejection andGraft-Versus-Host-Disease (GVHD) are T lymphocyte-dependent; inhibitionof T lymphocyte function is ameliorative.

Other diseases in which intervention of the immune and/or inflammatoryresponse have benefit are infectious disease including but not limitedto viral infection (including but not limited to AIDS, hepatitis A, B,C, D, E and herpes) bacterial infection, fungal infections, andprotozoal and parasitic infections (molecules (or derivatives/agonists)which stimulate the MLR can be utilized therapeutically to enhance theimmune response to infectious agents), diseases of immunodeficiency(molecules/derivatives/agonists) which stimulate the MLR can be utilizedtherapeutically to enhance the immune response for conditions ofinherited, acquired, infectious induced (as in HIV infection), oriatrogenic (i.e., as from chemotherapy) immunodeficiency, and neoplasia.

It has been demonstrated that some human cancer patients develop anantibody and/or T lymphocyte response to antigens on neoplastic cells.It has also been shown in animal models of neoplasia that enhancement ofthe immune response can result in rejection or regression of thatparticular neoplasm. Molecules that enhance the T lymphocyte response inthe MLR have utility in vivo in enhancing the immune response againstneoplasia. Molecules which enhance the T lymphocyte proliferativeresponse in the MLR (or small molecule agonists or antibodies thataffected the same receptor in an agonistic fashion) can be usedtherapeutically to treat cancer. Molecules that inhibit the lymphocyteresponse in the MLR also function in vivo during neoplasia to suppressthe immune response to a neoplasm; such molecules can either beexpressed by the neoplastic cells themselves or their expression can beinduced by the neoplasm in other cells. Antagonism of such inhibitorymolecules (either with antibody, small molecule antagonists or othermeans) enhances immune-mediated tumor rejection.

Additionally, inhibition of molecules with proinflammatory propertiesmay have therapeutic benefit in reperfusion injury; stroke; myocardialinfarction; atherosclerosis; acute lung injury; hemorrhagic shock; burn;sepsis/septic shock; acute tubular necrosis; endometriosis; degenerativejoint disease and pancreatis.

The compounds of the present invention, e.g., polypeptides orantibodies, are administered to a mammal, preferably a human, in accordwith known methods, such as intravenous administration as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation (intranasal,intrapulmonary) routes. Intravenous or inhaled administration ofpolypeptides and antibodies is preferred.

In immunoadjuvant therapy, other therapeutic regimens, suchadministration of an anti-cancer agent, may be combined with theadministration of the proteins, antibodies or compounds of the instantinvention. For example, the patient to be treated with a theimmunoadjuvant of the invention may also receive an anti-cancer agent(chemotherapeutic agent) or radiation therapy. Preparation and dosingschedules for such chemotherapeutic agents may be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in Chemotherapy Service Ed., M. C. Perry, Williams &Wilkins, Baltimore, Md. (1992). The chemotherapeutic agent may precede,or follow administration of the immunoadjuvant or may be givensimultaneously therewith. Additionally, an anti-estrogen compound suchas tamoxifen or an anti-progesterone such as onapristone (see, EP616812) may be given in dosages known for such molecules.

It may be desirable to also administer antibodies against other immunedisease associated or tumor associated antigens, such as antibodieswhich bind to CD20, CD11a, CD18, ErbB2, EGFR, ErbB3, ErbB4, or vascularendothelial factor (VEGF). Alternatively, or in addition, two or moreantibodies binding the same or two or more different antigens disclosedherein may be coadministered to the patient. Sometimes, it may bebeneficial to also administer one or more cytokines to the patient. Inone embodiment, the PRO87299 polypeptides are coadministered with agrowth inhibitory agent. For example, the growth inhibitory agent may beadministered first, followed by a PRO87299 polypeptide. However,simultaneous administration or administration first is alsocontemplated. Suitable dosages for the growth inhibitory agent are thosepresently used and may be lowered due to the combined action (synergy)of the growth inhibitory agent and the PRO87299 polypeptide.

For the treatment or reduction in the severity of immune relateddisease, the appropriate dosage of an a compound of the invention willdepend on the type of disease to be treated, as defined above, theseverity and course of the disease, whether the agent is administeredfor preventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the compound, and the discretion of theattending physician. The compound is suitably administered to thepatient at one time or over a series of treatments.

For example, depending on the type and severity of the disease, about 1μg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of polypeptide or antibody is aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

O. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials (e.g., comprising a PRO87299 molecule) useful forthe diagnosis or treatment of the disorders described above is provided.The article of manufacture comprises a container and an instruction.Suitable containers include, for example, bottles, vials, syringes, andtest tubes. The containers may be formed from a variety of materialssuch as glass or plastic. The container holds a composition which iseffective for diagnosing or treating the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agent in the composition is usually apolypeptide or an antibody of the invention. An instruction or label on,or associated with, the container indicates that the composition is usedfor diagnosing or treating the condition of choice. The article ofmanufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

P. Diagnosis and Prognosis of Immune Related Disease

Cell surface proteins, such as proteins which are overexpressed incertain immune related diseases, are excellent targets for drugcandidates or disease treatment. The same proteins along with secretedproteins encoded by the genes amplified in immune related disease statesfind additional use in the diagnosis and prognosis of these diseases.For example, antibodies directed against the protein products of genesamplified in multiple sclerosis, rheumatoid arthritis, or another immunerelated disease, can be used as diagnostics or prognostics.

For example, antibodies, including antibody fragments, can be used toqualitatively or quantitatively detect the expression of proteinsencoded by amplified or overexpressed genes (“marker gene products”).The antibody preferably is equipped with a detectable, e.g., fluorescentlabel, and binding can be monitored by light microscopy, flow cytometry,fluorimetry, or other techniques known in the art. These techniques areparticularly suitable, if the overexpressed gene encodes a cell surfaceprotein Such binding assays are performed essentially as describedabove.

In situ detection of antibody binding to the marker gene products can beperformed, for example, by immunofluorescence or immunoelectronmicroscopy. For this purpose, a histological specimen is removed fromthe patient, and a labeled antibody is applied to it, preferably byoverlaying the antibody on a biological sample. This procedure alsoallows for determining the distribution of the marker gene product inthe tissue examined. It will be apparent for those skilled in the artthat a wide variety of histological methods are readily available for insitu detection.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example 1 Cloning of PRO87299

An expressed sequence tag (EST) DNA database (Merck/WashingtonUniversity) was searched and an EST was identified which containeddomains of interest, specifically Immunoglobulin (Ig) domain(s) andImmuno Tyrosine Inhibition Motif(s) (ITIM). The search was performedusing the computer program BLAST or BLAST2 [Altschul et al., Methods inEnzymology, 266:460-480 (1996)] using as a comparison the domains ofinterest to a 6 frame translation of the sequences. Those comparisonsresulting in a BLAST score of 70 (or in some cases, 90) or greater thatdid not encode known proteins were clustered and if necessary, assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.).

Based on the sequence as described above, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO87299. Forward and reverse PCRprimers generally range from 20 to 30 nucleotides and are often designedto give a PCR product of about 100-1000 bp in length. The probesequences are typically 40-55 bp in length. In some cases, additionaloligonucleotides are synthesized when the consensus sequence is greaterthan about 1-1.5 kbp. In order to screen several libraries for afull-length clone, DNA from the libraries was screened by PCRamplification, as per Ausubel et al., Current Protocols in MolecularBiology, supra, with the PCR primer pair. A positive library was thenused to isolate clones encoding the gene of interest using the probeoligonucleotide and one of the primer pairs.

The oligonucleotide probes employed were as follows:

-   Forward primer: hBTig.EcoRI.F2 5′ TTGAATTCATGAAGACATTGCCTGCCATGC 3′    (SEQ ID NO: 3)-   Reverse primer: hBTig.BamHI.R2 5′    TTGGATCCTTAACTCCTCACACATATGGATGCATATTC 3′ (SEQ ID NO: 4)

A human blood cDNA library was used in cloning. The cDNA library used toisolate the cDNA clones were constructed by standard methods usingcommercially available reagents such as those from Invitrogen, SanDiego, Calif. The cDNA was primed with oligo dT containing a NotI site,linked with blunt to SalI hemikinased adaptors, cleaved with NotI, sizedappropriately by gel electrophoresis, and cloned in a definedorientation into a suitable cloning vector (such as pRKB or pRKD; pRK5Bis a precursor of pRK5D that does not contain the SfiI site; see, Holmeset al., Science, 253:1278-1280 (1991)) in the unique XhoI and NotIsites.

The entire nucleotide sequence of the clone, designated herein asDNA332467, is shown in FIG. 1 (SEQ ID NO: 1). The DNA332467 clonecontains a single open reading frame with an apparent translationalinitiation site at nucleotide positions 24-26 and a stop signal atnucleotide positions 891-893 (FIG. 1, SEQ ID NO:1). The predictedpolypeptide precursor is 289 amino acids long, has a calculatedmolecular weight of approximately 32781 daltons and an estimated pI ofapproximately 6.27. Analysis of the full-length PRO87299 sequence shownin FIG. 2 (SEQ ID NO:2) evidences the presence of a variety of importantpolypeptide domains as shown in FIG. 2, wherein the locations given forthose polypeptide domains are approximate as described.

An analysis of the current protein database, using the ALIGN-2 sequencealignment analysis of the full-length sequence shown in FIG. 2 (SEQ IDNO:2), evidenced sequence identity between the PRO87299 amino acidsequence and no known protein sequences.

Example 2 Microarray Analysis of Stimulated T-Cells

Nucleic acid microarrays, often containing thousands of gene sequences,are useful for identifying differentially expressed genes in diseasedtissues as compared to their normal counterparts. Using nucleic acidmicroarrays, test and control mRNA samples from test and control tissuesamples are reverse transcribed and labeled to generate cDNA probes. ThecDNA probes are then hybridized to an array of nucleic acids immobilizedon a solid support. The array is configured such that the sequence andposition of each member of the array is known. For example, a selectionof genes known to be expressed in certain disease states may be arrayedon a solid support. Hybridization of a labeled probe with a particulararray member indicates that the sample from which the probe was derivedexpresses that gene. If the hybridization signal of a probe from a test(in this instance, activated CD4+ T cells) sample is greater thanhybridization signal of a probe from a control (in this instance,non-stimulated CD4+ T cells) sample, the gene or genes overexpressed inthe test tissue are identified. The implication, of this result is thatan overexpressed protein in a test tissue is useful not only as adiagnostic marker for the presence of the disease condition, but also asa therapeutic target for treatment of the disease condition.

The methodology of hybridization of nucleic acids and microarraytechnology is well known in the art. In one example, the specificpreparation of nucleic acids for hybridization and probes, slides, andhybridization conditions are all detailed in PCT Patent ApplicationSerial No. PCT/US01/10482, filed on Mar. 30, 2001 and which is hereinincorporated by reference.

In this experiment, CD4+ T cells were purified from a single donor usingthe RossetteSep™ protocol from (Stem Cell Technologies, Vancouver BC)which contains anti-CD8, anti-CD16, anti-CD19, anti-CD36 and anti-CD56antibodies used to produce a population of isolated CD4+ T cells.Isolated CD4+ T cells were activated with an anti-CD3 antibody (used ata concentration that does not stimulate proliferation) together witheither ICAM-1 or anti-CD28 antibody. At 24 or 72 hours cells wereharvested, RNA extracted and analysis run on Affimax™ (Affymetrix Inc.Santa Clara, Calif.) microarrays. Non-stimulated (resting) cells wereharvested immediately after purification, and subjected to the sameanalysis. Genes were compared whose expression was upregulated at eitherof the two timepoints in activated vs. resting cells.

The result of these experiments, is that PRO87299 polypeptides of thepresent invention are significantly overexpressed in isolated CD4+ Tcells activated by anti-CD3/ICAM-1 and anti-CD3/anti-CD28 as compared toisolated resting CD4+ T cells. As described above, these datademonstrate that the PRO87299 polypeptides of the present invention areuseful not only as diagnostic markers for the presence of one or moreimmune disorders, but also serve as therapeutic targets for thetreatment of those immune disorders.

Example 3 PRO87299 in Lymphoma

Lymphoma is the 6th most common malignancy in the United States. Therewere an estimated 43,000 new cases of lymphoma in the United States in1990. Non-Hodgkin's lymphoma accounts for the majority of cases, withHodgkin's lymphoma cases a distant second. The incidence ofNon-Hodgkin's lymphoma progressively increases with age. But inHodgkin's disease, there is a high incidence in patients ages 20-30, aplateau between 30-55 and another rise after age 55. Males are higher atrisk for both Hodgkin's disease and non-Hodgkin's lymphoma than females.The major clinical manifestation of malignant lymphoma is swelling ofthe lymph node and symptoms include fever, malaise, and weight loss.Common primary sites of lymphoma include supraclavicular, axillary,mediastinal, periaortic, cervical, and inguinal lymph nodes. Lymphomaalso has the potential to metastasize to other organs.

Hodgkin's disease was first described by Thomas Hodgkin in 1832.Hodgkin's disease is an unrestricted proliferation of a lymphoid cellwhich becomes larger, with abundant pale cytoplasm and two or more ovallobulated nuclei containing large nucleoli. Cells of this appearance areknown as Reed-Sternberg cells. Reed-Sternberg cells are important forthe diagnosis of Hodgkin's disease, but their presence alone is notsufficient for diagnosis. Hodgkin's disease is distinct from non-Hodgkinlymphoma by cell type, lymph node histology, and by the symptomatology,such as fever. Hodgkin's disease generally presents as enlargement of asingle group of peripheral lymph nodes, and may involve contiguousnodes, but is infrequently extranodal. The cause of Hodgkin's disease isunknown, but prior Epstein Barr Virus infection and bcl-2 translocationsare associated with the development of Hodgkin disease.

The non-Hodgkin's lymphomas are neoplasms of the immune system arisingin lymph nodes, but are differentiated from Hodgkin's disease by factorssuch as the cell type and the symptomatology exhibited by the patient.Most non-Hodgkin's lymphomas are of B cell phenotype and are positivefor the markers CD19 and CD20. A smaller number are T cell lymphomas andare positive for the markers CD2 and CD3.

A proprietary database containing gene expression information(GeneExpress®, Gene Logic Inc., Gaithersburg, Md.) was analyzed in anattempt to identify if PRO87299 polypeptide (and its encoding nucleicacids) is significantly upregulated in lymphoma as compared to normallymph tissues. Specifically, analysis of the GeneExpress® database wasconducted using either software available through Gene Logic Inc.,Gaithersburg, Md., for use with the GeneExpress® database or withproprietary software written and developed at Genentech, Inc. for usewith the GeneExpress® database. The rating of positive hits in theanalysis is based upon several criteria including, for example, tissuespecificity, tumor specificity and expression level in normal essentialand/or normal proliferating tissues. The result is that PRO87299 doesevidence high expression in lymphoma as compared to other tumors and/ornormal tissues and optionally relatively low expression in normalessential and/or normal proliferating tissues.

Example 4 PRO87299 in Inflammatory Bowel Disease

In this experiment, a microarray assay was used to find genes that areoverexpressed in IBD as compared to normal bowel tissue. Biopsies frompatients with IBD were obtained. For each IBD patient, samples weretaken from disease (either UC or Crohn's) tissue and from healthy bowel,so that expression patterns could be better compared. All samples werestored at −70° C. until ready for RNA isolation. The biopsies werehomogenized in 600 ul of RLT buffer (+BME) and RNA was isolated usingQiagen™ Rneasy Mini columns (Qiagen) with on-column DNase treatmentfollowing the manufacturer's guidelines. Following RNA isolation, RNAwas quantitated using RiboGreen™ (Molecular Probes) following themanufacturer's guidelines and checked on agarose gels for integrity.Appropriate amounts of RNA were labeled for microarray analysis andsamples were run on proprietary Genentech microarray and Affymetrics™microarrays. Genes were compared whose expression was upregulated in IBDtissue vs normal bowel, matching biopsies from normal bowel and IBDtissue from the same patient. The results of this experiment showed thatPRO87299 has been identified as being significantly overexpressed inCrohn's Disease samples as compared to normal bowel tissue.

Example 5 Expression of PRO87299 in NK Cells

Natural killer (NK) cells are an important effector cell of the innateimmune system. They are specialized to effect killing against host cellsthat have either been infected by viruses, parasites or that have becomecancerous. Phenotypically, NK cells are large granular lymphocytes thatconstitute ˜2% of the circulating lymphocyte population. They arecommonly identified by cell surface expression of CD56 and CD16 Theymature in the bone marrow from a CD34+precursor cell that they sharewith T cells. The mature NK cell, shares expression of CD8, cytolyticmachinery, and some KIRs, with T cells, but remains distinct from Tcells by the lack of CD3 and the T cell receptors. Like cytotoxic Tcells, they contain granules filled with pore forming protein,cytotoxins, serine esterases and proteoglycans that mediate lysis oftarget cells. Both cytotoxic T cells and NK cells kill on contact bybinding to their targets and delivering their lethal burst of chemicalsthat produces holes in the target cell's membrane. Unlike cytotoxic Tcells, NK cells do not need to recognize a specific antigen beforeinitiating lysis. Rather, NK cell activation can be mediated by growthfactors and cytokines (in particular, IL-2, IL-12 and IL-15 have beenshown to mediate proliferative and cytotoxic activities or by a delicatebalance between two classes of NK cell receptors, one that activates thecells, and another that inhibits. Killer Ig-like receptors (KIRs) are NKcell receptors that transmit an inhibitory signal if they encounterclass I MHC molecules on a cell surface. This is important for killingof both cancerous cells and virally infected cells. Because virusesoften suppress class I MHC expression in cells they infect, thevirus-infected cell becomes susceptible to killing by NK cells.Likewise, cancer cells have reduced or no class I MHC expression andthey, too, become susceptible to killing by NK cells. Naturalcytotoxicity receptors (NCRs) constitute a family of activatingreceptors on NK cells. In some effector-target systems, the surfacedensity of NCRs correlates with the cytolytic activity of the NK cells,while in other systems killing requires cooperation between NCR, anotheractivating receptor NKG2D and its adaptor polypeptide DAP10.Additionally, the strength of the signals can be influenced byengagement of coreceptors such as 2B4 and NTB-A. The ligands for NCRsand NKG2D, hemoglutanins and MICA, MICB respectively are not expressedby most normal cells, but are induced in most tumor cell lines.Expression of the ligands by tumor cells triggers a dramatic immuneresponse resulting in tumor cell rejection. Activation of NK cells withIL-15 or IL-12 have been shown to induce both cytotoxic andproliferative effects. Junctinal adhesion molecule 2 (JAM2) has beenshown to bind to NK cells and has been hypothesized to play a role inlymphocyte extravasation to sites of inflammation.

Therefore, a DNA microarray experiment comparing differential expressionof genes from these three modes of activation versus resting NK cellshas the potential to reveal novel genes or novel gene associations withNK cell activity. Therapeutic antibodies, peptides or small moleculescould be developed to target specific genes revealed by thesemicroarrays for the treatment of immune mediated inflammatory diseasesand malignancies. Peripheral blood NK cells were isolated fromleukopacks by negative selection using the NK cell isolation kit withthe MACS™ magnetic cell sorting system (Miltenyi Biotec). Cell puritywas confirmed by staining with PE anti-CD56 for FACS analysis. Purity ofcell preps ranged from 89% to 96%. Cell culture: Set up in-vitrocultures in 6 well plates 5 ml cultures/well. Media: RPMI 1640, 10% heatinactivated FBS, 100 units/mL of Penicillin, 100 mg/mL of streptomycin,2 mM L-glutamine, and 5.5×10−5 Beta-mercaptoethanol. Experimentaltreatments: Time 0 hrs, Untreated CD56(+) cells. Time 16 hrs. Untreated,IL2 (10 nM), IL15(10 nM), JAM-IT (10 nM) stimulated. Activation of NKcells was monitored by FACS for cell surface expression of CD56 andCD69. In this series of experiments it was determined that PRO87299 isexpressed in CD56+NK cells, when compared with normal resting NK cells.

Example 6 Use of PRO87299 as a Hybridization Probe

The following method describes use of a nucleotide sequence encodingPRO87299 as a hybridization probe.

DNA comprising the coding sequence of full-length or mature PRO87299 asdisclosed herein is employed as a probe to screen for homologous DNAs(such as those encoding naturally-occurring variants of PRO87299) inhuman tissue cDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled PRO87299-derived probe to the filters is performed in asolution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodium pyrophosphate,50 mM sodium phosphate, pH 6.8, 2× Denhardt's solution, and 10% dextransulfate at 42° C. for 20 hours. Washing of the filters is performed inan aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence PRO87299 can then be identified usingstandard techniques known in the art.

Example 7 Expression of PRO87299 in E. coli

This example illustrates preparation of an unglycosylated form ofPRO87299 by recombinant expression in E. coli.

The DNA sequence encoding PRO87299 is initially amplified using selectedPCR primers. The primers should contain restriction enzyme sites whichcorrespond to the restriction enzyme sites on the selected expressionvector. A variety of expression vectors may be employed. An example of asuitable vector is pBR322 (derived from E. coli; see Bolivar et al.,Gene, 2:95 (1977)) which contains genes for ampicillin and tetracyclineresistance. The vector is digested with restriction enzyme anddephosphorylated. The PCR amplified sequences are then ligated into thevector. The vector will preferably include sequences which encode for anantibiotic resistance gene, a trp promoter, a polyhis leader (includingthe first six STII codons, polyhis sequence, and enterokinase cleavagesite), the PRO87299 coding region, lambda transcriptional terminator,and an argU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO87299 protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

PRO87299 may be expressed in E. coli in a poly-His tagged form, usingthe following procedure. The DNA encoding PRO87299 is initiallyamplified using selected PCR primers. The primers will containrestriction enzyme sites which correspond to the restriction enzymesites on the selected expression vector, and other useful sequencesproviding for efficient and reliable translation initiation, rapidpurification on a metal chelation column, and proteolytic removal withenterokinase. The PCR-amplified, poly-His tagged sequences are thenligated into an expression vector, which is used to transform an E. colihost based on strain 52 (W3110 fuhA(tonA) Ion galE rpoHts(htpRts)clpP(lacIq). Transformants are first grown in LB containing 50 mg/mlcarbenicillin at 30° C. with shaking until an O.D.600 of 3-5 is reached.Cultures are then diluted 50-100 fold into CRAP media (prepared bymixing 3.57 g (NH₄)₂SO₄, 0.71 g sodium citrate·2H2O, 1.07 g KCl, 5.36 gDifco yeast extract, 5.36 g Sheffield hycase SF in 500 μL water, as wellas 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grownfor approximately 20-30 hours at 30° C. with shaking. Samples areremoved to verify expression by SDS-PAGE analysis, and the bulk cultureis centrifuged to pellet the cells. Cell pellets are frozen untilpurification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant is diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 ml QiagenNi-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

The proteins are refolded by diluting the sample slowly into freshlyprepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refoldingvolumes are chosen so that the final protein concentration is between 50to 100 micrograms/ml. The refolding solution is stirred gently at 4° C.for 12-36 hours. The refolding reaction is quenched by the addition ofTFA to a final concentration of 0.4% (pH of approximately 3). Beforefurther purification of the protein, the solution is filtered through a0.22 micron filter and acetonitrile is added to 2-10% finalconcentration. The refolded protein is chromatographed on a Poros R1/Hreversed phase column using a mobile buffer of 0.1% TFA with elutionwith a gradient of acetonitrile from 10 to 80%. Aliquots of fractionswith A280 absorbance are analyzed on SDS polyacrylamide gels andfractions containing homogeneous refolded protein are pooled. Generally,the properly refolded species of most proteins are eluted at the lowestconcentrations of acetonitrile since those species are the most compactwith their hydrophobic interiors shielded from interaction with thereversed phase resin. Aggregated species are usually eluted at higheracetonitrile concentrations. In addition to resolving misfolded forms ofproteins from the desired form, the reversed phase step also removesendotoxin from the samples.

Fractions containing the desired folded PRO87299 polypeptide are pooledand the acetonitrile removed using a gentle stream of nitrogen directedat the solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtrationusing G25 Superfine (Pharmacia) resins equilibrated in the formulationbuffer and sterile filtered.

The PRO87299 polypeptides disclosed herein were successfully expressedas described above.

Example 8 Expression of PRO87299 in Mammalian Cells

This example illustrates preparation of a potentially glycosylated formof PRO87299 by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the PRO87299 DNA is ligated intopRK5 with selected restriction enzymes to allow insertion of thePRO87299 DNA using ligation methods such as described in Sambrook etal., supra. The resulting vector is called pRK5-PRO87299.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μgpRK5-PRO87299 DNA is mixed with about 1 μg DNA encoding the VA RNA gene[Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added,dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄,and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of PRO87299 polypeptide. The cultures containing transfectedcells may undergo further incubation (in serum free medium) and themedium is tested in selected bioassays.

In an alternative technique, PRO87299 may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg pRK5-PRO87299 DNA isadded. The cells are first concentrated from the spinner flask bycentrifugation and washed with PBS. The DNA-dextran precipitate isincubated on the cell pellet for four hours. The cells are treated with20% glycerol for 90 seconds, washed with tissue culture medium, andre-introduced into the spinner flask containing tissue culture medium, 5μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about fourdays, the conditioned media is centrifuged and filtered to remove cellsand debris. The sample containing expressed PRO87299 can then beconcentrated and purified by any selected method, such as dialysisand/or column chromatography.

In another embodiment, PRO87299 can be expressed in CHO cells. ThepRK5-PRO87299 can be transfected into CHO cells using known reagentssuch as CaPO₄ or DEAE-dextran. As described above, the cell cultures canbe incubated, and the medium replaced with culture medium (alone) ormedium containing a radiolabel such as ³⁵S-methionine. After determiningthe presence of PRO87299 polypeptide, the culture medium may be replacedwith serum free medium. Preferably, the cultures are incubated for about6 days, and then the conditioned medium is harvested. The mediumcontaining the expressed PRO87299 can then be concentrated and purifiedby any selected method.

Epitope-tagged PRO87299 may also be expressed in host CHO cells. ThePRO87299 may be subcloned out of the pRK5 vector. The subclone insertcan undergo PCR to fuse in frame with a selected epitope tag such as apoly-his tag into a Baculovirus expression vector. The poly-his taggedPRO87299 insert can then be subcloned into a SV40 promoter/enhancercontaining vector containing a selection marker such as DHFR forselection of stable clones. Finally, the CHO cells can be transfected(as described above) with the SV40 promoter/enhancer containing vector.Labeling may be performed, as described above, to verify expression. Theculture medium containing the expressed poly-His tagged PRO87299 canthen be concentrated and purified by any selected method, such as byNi²⁺-chelate affinity chromatography.

PRO87299 may also be expressed in CHO and/or COS cells by a transientexpression procedure or in CHO cells by another stable expressionprocedure.

Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins are fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or is a poly-His tagged form.

Following PCR amplification, the respective DNAs are subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are constructed to have compatiblerestriction sites 5′ and 3′ of the DNA of interest to allow theconvenient shuttling of cDNA's. The vector used expression in CHO cellsis as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Quiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁻⁷ cells are frozen in an ampule for furthergrowth and production as described below.

The ampules containing the plasmid DNA are thawed by placement intowater bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 mL of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL, of selective media. After 1-2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2-3 days,250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3 L production spinner isseeded at 1.2×10⁶ cells/mL. On day 0, pH is determined. On day 1, thespinner is sampled and sparging with filtered air is commenced. On day2, the spinner is sampled, the temperature shifted to 33° C., and 30 mLof 500 g/L glucose and 0.6 mL of 10% antifoam (e.g., 35%polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion)taken. Throughout the production, the pH is adjusted as necessary tokeep it at around 7.2. After 10 days, or until the viability droppedbelow 70%, the cell culture is harvested by centrifugation and filteringthrough a 0.22 μm filter. The filtrate was either stored at 4° C. orimmediately loaded onto columns for purification.

For the poly-His tagged constructs, the proteins are purified using aNi-NTA column (Qiagen). Before purification, imidazole is added to theconditioned media to a concentration of 5 mM. The conditioned media ispumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5ml/min. at 4° C. After loading, the column is washed with additionalequilibration buffer and the protein eluted with equilibration buffercontaining 0.25 M imidazole. The highly purified protein is subsequentlydesalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column andstored at −80° C.

Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μl of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

Many of the PRO87299 polypeptides disclosed herein were successfullyexpressed as described above.

Example 9 Expression of PRO87299 in Yeast

The following method describes recombinant expression of PRO87299 inyeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO87299 from the ADH2/GAPDH promoter. DNAencoding PRO87299 and the promoter is inserted into suitable restrictionenzyme sites in the selected plasmid to direct intracellular expressionof PRO87299. For secretion, DNA encoding PRO87299 can be cloned into theselected plasmid, together with DNA encoding the ADH2/GAPDH promoter, anative PRO87299 signal peptide or other mammalian signal peptide, or,for example, a yeast alpha-factor or invertase secretory signal/leadersequence, and linker sequences (if needed) for expression of PRO87299.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant PRO87299 can subsequently be isolated and purified byremoving the yeast cells from the fermentation medium by centrifugationand then concentrating the medium using selected cartridge filters. Theconcentrate containing PRO87299 may further be purified using selectedcolumn chromatography resins.

Many of the PRO87299 polypeptides disclosed herein were successfullyexpressed as described above.

Example 10 Expression of PRO87299 in Baculovirus-Infected Insect Cells

The following method describes recombinant expression of PRO87299 inBaculovirus-infected insect cells.

The sequence coding for PRO87299 is fused upstream of an epitope tagcontained within a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thesequence encoding PRO87299 or the desired portion of the coding sequenceof PRO87299 such as the sequence encoding the extracellular domain of atransmembrane protein or the sequence encoding the mature protein if theprotein is extracellular is amplified by PCR with primers complementaryto the 5′ and 3′ regions. The 5′ primer may incorporate flanking(selected) restriction enzyme sites. The product is then digested withthose selected restriction enzymes and subcloned into the expressionvector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged PRO87299 can then be purified, for example, byNi²⁺-chelate affinity chromatography as follows. Extracts are preparedfrom recombinant virus-infected Sf9 cells as described by Rupert et al.,Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspendedin sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl₂; 0.1 mM EDTA;10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 secondson ice. The sonicates are cleared by centrifugation, and the supernatantis diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni²⁺-NTAagarose column (commercially available from Qiagen) is prepared with abed volume of 5 mL, washed with 25 mL of water and equilibrated with 25mL of loading buffer. The filtered cell extract is loaded onto thecolumn at 0.5 mL per minute. The column is washed to baseline A₂₈₀ withloading buffer, at which point fraction collection is started. Next, thecolumn is washed with a secondary wash buffer (50 mM phosphate; 300 mMNaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.After reaching A₂₈₀ baseline again, the column is developed with a 0 to500 mM Imidazole gradient in the secondary wash buffer. One mL fractionsare collected and analyzed by SDS-PAGE and silver staining or Westernblot with Ni²⁺-NTA-conjugated to alkaline phosphatase (Qiagen).Fractions containing the eluted His₁₀-tagged PRO87299 are pooled anddialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) PRO87299can be performed using known chromatography techniques, including forinstance, Protein A or protein G column chromatography.

Many of the PRO87299 polypeptides disclosed herein were successfullyexpressed as described above.

Example 11 Preparation of Antibodies that Bind PRO87299

This example illustrates preparation of monoclonal antibodies which canspecifically bind PRO87299.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified PRO87299, fusion proteins containingPRO87299, and cells expressing recombinant PRO87299 on the cell surface.Selection of the immunogen can be made by the skilled artisan withoutundue experimentation.

Mice, such as Balb/c, are immunized with the PRO87299 immunogenemulsified in complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-PRO87299 antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO87299. Three to four days later, the mice are sacrificedand the spleen cells are harvested. The spleen cells are then fused(using 35% polyethylene glycol) to a selected murine myeloma cell linesuch as P3X63AgU.1, available from ATCC, No. CRL 1597. The fusionsgenerate hybridoma cells which can then be plated in 96 well tissueculture plates containing HAT (hypoxanthine, aminopterin, and thymidine)medium to inhibit proliferation of non-fused cells, myeloma hybrids, andspleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstPRO87299. Determination of “positive” hybridoma cells secreting thedesired monoclonal antibodies against PRO87299 is within the skill inthe art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-PRO87299monoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 12 Purification of PRO87299 Polypeptides Using SpecificAntibodies

Native or recombinant PRO87299 polypeptides may be purified by a varietyof standard techniques in the art of protein purification. For example,pro-PRO87299 polypeptide, mature PRO87299 polypeptide, or pre-PRO87299polypeptide is purified by immunoaffinity chromatography usingantibodies specific for the PRO87299 polypeptide of interest. Ingeneral, an immunoaffinity column is constructed by covalently couplingthe anti-PRO87299 polypeptide antibody to an activated chromatographicresin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such an immunoaffinity column is utilized in the purification ofPRO87299 polypeptide by preparing a fraction from cells containingPRO87299 polypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble PRO87299polypeptide containing a signal sequence may be secreted in usefulquantity into the medium in which the cells are grown.

A soluble PRO87299 polypeptide-containing preparation is passed over theimmunoaffinity column, and the column is washed under conditions thatallow the preferential absorbance of PRO87299 polypeptide (e.g., highionic strength buffers in the presence of detergent). Then, the columnis eluted under conditions that disrupt antibody/PRO87299 polypeptidebinding (e.g., a low pH buffer such as approximately pH 2-3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), andPRO87299 polypeptide is collected.

Example 13 Drug Screening

This invention is particularly useful for screening compounds by usingPRO87299 polypeptides or binding fragment thereof in any of a variety ofdrug screening techniques. The PRO87299 polypeptide or fragment employedin such a test may either be free in solution, affixed to a solidsupport, borne on a cell surface, or located intracellularly. One methodof drug screening utilizes eukaryotic or prokaryotic host cells whichare stably transformed with recombinant nucleic acids expressing thePRO87299 polypeptide or fragment. Drugs are screened against suchtransformed cells in competitive binding assays. Such cells, either inviable or fixed form, can be used for standard binding assays. One maymeasure, for example, the formation of complexes between PRO87299polypeptide or a fragment and the agent being tested. Alternatively, onecan examine the diminution in complex formation between the PRO87299polypeptide and its target cell or target receptors caused by the agentbeing tested.

Thus, the present invention provides methods of screening for drugs orany other agents which can affect a PRO87299 polypeptide-associateddisease or disorder. These methods comprise contacting such an agentwith an PRO87299 polypeptide or fragment thereof and assaying (i) forthe presence of a complex between the agent and the PRO87299 polypeptideor fragment, or (ii) for the presence of a complex between the PRO87299polypeptide or fragment and the cell, by methods well known in the art.In such competitive binding assays, the PRO87299 polypeptide or fragmentis typically labeled. After suitable incubation, free PRO87299polypeptide or fragment is separated from that present in bound form,and the amount of free or uncomplexed label is a measure of the abilityof the particular agent to bind to PRO87299 polypeptide or to interferewith the PRO87299 polypeptide/cell complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to a polypeptide and isdescribed in detail in WO84/03564, published on Sep. 13, 1984. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. As applied to a PRO87299 polypeptide, the peptide testcompounds are reacted with PRO87299 polypeptide and washed. BoundPRO87299 polypeptide is detected by methods well known in the art.Purified PRO87299 polypeptide can also be coated directly onto platesfor use in the aforementioned drug screening techniques. In addition,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding PRO87299polypeptide specifically compete with a test compound for binding toPRO87299 polypeptide or fragments thereof. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with PRO87299 polypeptide.

Example 14 Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptide of interest (i.e., a PRO87299polypeptide) or of small molecules with which they interact, e.g.,agonists, antagonists, or inhibitors. Any of these examples can be usedto fashion drugs which are more active or stable forms of the PRO87299polypeptide or which enhance or interfere with the function of thePRO87299 polypeptide in vivo (c.f., Hodgson, Bio/Technology, 9: 19-21(1991)).

In one approach, the three-dimensional structure of the PRO87299polypeptide, or of a PRO87299 polypeptide-inhibitor complex, isdetermined by x-ray crystallography, by computer modeling or, mosttypically, by a combination of the two approaches. Both the shape andcharges of the PRO87299 polypeptide must be ascertained to elucidate thestructure and to determine active site(s) of the molecule. Less often,useful information regarding the structure of the PRO87299 polypeptidemay be gained by modeling based on the structure of homologous proteins.In both cases, relevant structural information is used to designanalogous PRO87299 polypeptide-like molecules or to identify efficientinhibitors. Useful examples of rational drug design may includemolecules which have improved activity or stability as shown by Braxtonand Wells, Biochemistry, 31:7796-7801 (1992) or which act as inhibitors,agonists, or antagonists of native peptides as shown by Athauda et al.,J. Biochem. 113:742-746 (1993).

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amounts of the PRO87299polypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, knowledge of the PRO87299polypeptide amino acid sequence provided herein will provide guidance tothose employing computer modeling techniques in place of or in additionto x-ray crystallography.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. An isolated polypeptide comprising an amino acid sequence having atleast 95% amino acid sequence identity over the full length of the aminoacid sequence of SEQ ID NO:2, wherein the polypeptide comprises an Igdomain and an ITIM domain, and wherein the polypeptide is capable ofinducing the production of antibodies that detect overexpression of theprotein of SEQ ID NO:2 in activated CD4+ T cells.
 2. A chimeric moleculecomprising a polypeptide according to claim 1 fused to a heterologousamino acid sequence.
 3. The chimeric molecule of claim 2, wherein saidheterologous amino acid sequence is an epitope tag sequence or an Fcregion of an immunoglobulin.
 4. The isolated polypeptide of claim 1comprising an amino acid sequence having at least 99% amino acidsequence identity over the full length of the amino acid sequence of SEQID NO:2.
 5. The isolated polypeptide of claim 1 comprising the aminoacid sequence of SEQ ID NO:2.
 6. An isolated polypeptide comprising anamino acid sequence having at least 95% amino acid sequence identityover the full length of the extracellular domain of SEQ ID NO:2, whereinthe polypeptide comprises an Ig domain, and wherein the polypeptide iscapable of inducing the production of antibodies that detectoverexpression of the protein of SEQ ID NO:2 in activated CD4+ T cells.7. The isolated polypeptide of claim 6 comprising an amino acid sequencehaving at least 99% amino acid sequence identity over the full length ofthe extracellular domain of SEQ ID NO:2.
 8. The isolated polypeptide ofclaim 6 comprising the amino acid sequence of the extracellular domainof SEQ ID NO:2.
 9. A chimeric molecule comprising a polypeptideaccording to claim 6 fused to a heterologous amino acid sequence. 10.The chimeric molecule of claim 9, wherein said heterologous amino acidsequence is an epitope tag sequence or an Fc region of animmunoglobulin.